Cas proteins and guide rna systems for genome editing

ABSTRACT

Compositions and methods are provided for restoring function to a non-functional gene product in the genome of a cell. The methods and compositions employ a guide polynucleotide/Cas endonuclease system to restore function to a non-functional gene product and to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. The present disclosure also describes methods for modifying a nucleotide sequence in the genome of a cell using a restored functional selectable marker, as well as methods for editing a nucleotide sequence in the genome a cell without introducing a polynucleotide modification template into said cell. Compositions and methods are also provided for DNA free delivery of Cas endonucleases, sgRNAs and guide RNA/Cas complexes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/765,039, filed 30 Mar. 2018, which is the 371 national stage entry of International Application Number PCT/US2016/057279 filed on 17 Oct. 2016, which claims the benefit of U.S. Provisional Application No. 62/243,719, filed Oct. 20, 2015, U.S. Provisional Application No. 62/309,033, filed Mar. 16, 2016 and U.S. Provisional Application No. 62/359,254, filed Jul. 7, 2016, each of which is incorporated herein in their entirety by reference.

FIELD

The disclosure relates to the field of molecular biology, in particular, to methods for altering the genome of a cell.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named BB2533USPCN_SeqListing_ST25 created on 16 Jul. 2018 and having a size of 189,170 bytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND

Recombinant DNA technology has made it possible to insert DNA sequences at targeted genomic locations and/or modify (edit) specific endogenous chromosomal sequences, thus altering the organism's phenotype. Site-specific integration techniques, which employ site-specific recombination systems, as well as other types of recombination technologies, have been used to generate targeted insertions of genes of interest in a variety of organism. Genome-editing techniques such as designer zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), or homing meganucleases, are available for producing targeted genome perturbations, but these systems tends to have a low specificity and employ designed nucleases that need to be redesigned for each target site, which renders them costly and time-consuming to prepare.

Although several approaches have been developed to target a specific site for modification in the genome of an organism, there still remains a need for new genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the genome of an organism.

BRIEF SUMMARY

Compositions and methods are provided for restoring function to a non-functional gene product in the genome of a cell. The methods and compositions employ a guide polynucleotide/Cas endonuclease system to restore function to a non-functional gene product and to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. The present disclosure also describes methods for modifying a nucleotide sequence in the genome of a cell using a restored functional selectable marker, as well as methods for editing a nucleotide sequence in the genome a cell without introducing a polynucleotide modification template into said cell. Compositions and methods are also provided for DNA free delivery of Cas endonucleases, guide RNAs and guide RNA/Cas complexes by introducing the components of the complex via mRNA molecules (guide RNA, Cas endonuclease) or protein molecules (Cas endonuclease) or by directly introducing the guide RNA/Cas ribonucleotide-protein complex (RGEN) itself into the cell.

In one embodiment of the disclosure, the method comprises a method for restoring function to a non-functional gene product in the genome of a cell, the method comprising introducing a guide RNA/Cas endonuclease complex into a cell comprising a disrupted gene in its genome, wherein said complex creates a double strand break, wherein said disrupted gene does not encode a functional gene product, wherein said disrupted gene is restored without the use of a polynucleotide modification template to a non-disrupted gene capable of encoding said functional gene product. The disrupted gene can comprise a base pair deletion of the 4th nucleotide upstream (5′) of a PAM sequence when compared to its corresponding non-disrupted gene, wherein said base pair deletion creates an amino acid frameshift in the gene product of the disrupted gene thereby rendering the gene product of the disrupted gene non-functional. The base pair deletion can be located at the first, second or third nucleotide of a codon sequence. The restoration can be accomplished by Non-Homologous-End-Joining (NHEJ) resulting in the insertion of a single base at the double strand break site, or can be accomplished by the insertion of a single base at the double strand break site without the use of Homologous Recombination or Homology Directed Repair

In one embodiment of the disclosure, the method comprises a method for modifying a nucleotide sequence in the genome of a cell, the method comprising: introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein. The introducing and selection step does not comprise the introduction of a selectable marker gene, such as a recombinant DNA construct comprising a selectable marker gene. The disrupted selectable marker gene can be any disrupted marker gene including a disrupted visible marker gene. The modification in the targeted nucleotide sequence can be selected from the group consisting of an insertion of at least one nucleotide, a deletion of at least one nucleotide, or a substitution of at least one nucleotide in said target site. The method can further comprise introducing a polynucleotide modification template into said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence, or a donor DNA wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site.

In one embodiment of the disclosure, the method comprises a method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising: a) introducing into at least one cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence; b) selecting a cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; and, c) introducing into a cell of (b) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and insert a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template.

In one embodiment of the disclosure, the method comprises a method for editing a nucleotide sequence in the genome of a plant without the use of a polynucleotide modification template or donor DNA, the method comprising: a) introducing into at least one plant cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence; b) selecting a plant cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; c) regenerating a plant from the plant cell of (b); d) introducing into a cell from the plant of (c) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and inserting a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template; and, e) optimally, selecting a cell comprising the nucleotide insertion of (d).

The guide RNA and Cas endonuclease protein forming the guide RNA/Cas endonuclease complex can be introduced into the cell directly as RNA and protein, respectively, or as a ribonucleotide-protein complex. The components of the guide RNA/Cas endonuclease complex can be introduced as mRNA molecules encoding the Cas endonuclease protein and as RNA comprising the guide RNA, or as recombinant DNA molecules encoding the guide RNA and the Cas endonuclease protein.

In one embodiment of the disclosure, the method comprises a DNA free method of delivering a guide RNA/Cas endonuclease complex into a cell, the method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotide-protein and matrix to bind and form a ribonucleotide-protein-matrix complex; and, introducing said ribonucleotide-protein-matrix complex into said cell.

In one embodiment of the disclosure, the method comprises a DNA free method of delivering guide RNA/Cas endonuclease components into a cell, the method comprising introducing at least one guide RNA molecule and at least one Cas endonuclease protein into a cell, and growing said cell under suitable conditions to allow said guide RNA and said Cas endonuclease protein to form a complex inside said cell.

The DNA free method can further comprise introducing a polynucleotide template, wherein said polynucleotide modification template comprises at least one nucleotide modification of a nucleotide sequence in the genome of said cell, wherein said at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). The DNA free method can also further comprise introducing a donor DNA, wherein said donor DNA comprises at least one polynucleotide of interest. The introduction into the cell can be via a delivery system selected from the group consisting of particle mediated delivery, whisker mediated delivery, cell-penetrating peptide mediated delivery, electroporation, PEP-mediated transfection and nanoparticle mediated delivery.

Cells include human, non-human, animal, archaea, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cell.

Also provided are nucleic acid constructs, cells, plants, progeny plants, microorganisms, explants, seeds and grain produced by the methods described herein. Additional embodiments of the methods and compositions of the present disclosure are shown herein.

BRIEF DESCRIPTION OF THE DRAWINGS AND THE SEQUENCE LISTING

The disclosure can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application. The sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§ 1.821-1.825. The sequence descriptions contain the three letter codes for amino acids as defined in 37 C.F.R. §§ 1.821-1.825, which are incorporated herein by reference.

FIGURES

FIG. 1 depicts an alignment and count of the top 10 most frequent NHEJ mutations induced by the maize optimized guide RNA/Cas endonuclease system described herein. The mutations were identified by deep sequencing. The reference sequence (SEQ ID NO: 48) represents the unmodified locus with each target site shown in bold. The PAM sequence (grey) and expected site of cleavage (arrow) are also indicated. Deletions or insertions as a result of imperfect NHEJ are shown by a “-” or an italicized underlined nucleotide, respectively. The reference and mutations 1-10 of the target site correspond to SEQ ID NOs: 49-58, respectively. In maize, for the majority of target sites, the most prevalent type of mutation generated by Cas9-gRNA system is a single nucleotide insertion (≥60%) (count shown as 16,861).

FIG. 2 depicts partial nucleotide sequences ((SEQ ID NOs: 59, 61, 63) and partial amino acid sequences (SEQ ID NOs: 60, 62, 64) of the ALS2 gene and two editing repair templates (Oligo1 and Oligo2); modified nucleotides are underlined and the codon sequence targeted for gene editing (Pro to Ser) is boxed.

FIG. 3 depicts maize plants having an edited ALS2 allele for resistance to chlorsulfuron (left) and wild type plants (right). Four-week old plants were sprayed with chlorsulfuron (100 mg/L). Plants are shown three weeks after the treatment.

FIG. 4A-4C shows a schematic of a fragment of the ALS2 gene (SEQ ID NOs: 65, 67, 69) selected for modification and use of ALS2 as a selectable marker. The encoded amino acid sequences are shown below each nucleotide sequence. (SEQ ID NOs: 66, 68). FIG. 4A: A single nucleotide (G) in position 165 (bold and underlined) can be removed in order to generate a specific knock-out version of the edited for chlorsulfuron resistance ALS2 gene. FIG. 4B depicts the new nucleotide sequence with a single nucleotide deletion (G removed) resulting in the translational frame shift and ALS2 gene knock-out. FIG. 4C: The ALS2 gene function and chlorsulfuron resistance are restored through insertion of a single nucleotide (N, bold and underlined) during the process of DSB repair via NHEJ pathway.

FIG. 5A-5B. Re-activation of inactivated ALS2P165S as selectable marker. FIG. 5A (SEQ ID NO: 70). A design of ALS 2P165S gene containing upstream out-of-frame translational start codon located 3 nucleotides 5′ of PAM. Initiation of translation at the first AUG (depicted by arrow below sequence) encodes a 4 amino acid polypeptide which prevents the initiation of translation start codon of ALS2 (grey letters). FIG. 5B (SEQ ID NO: 71). Single nucleotide insertion (C, A or T) or deletion (or any combination) that results in the loss of the upstream AUG allows initiation of translation at the start codon of ALS2 (depicted by arrow below sequence) restoring translation of the full-length ALS2P165S herbicide resistance gene.

FIG. 6A-6C shows a schematic of a fragment of polynucleotide of interest (SEQ ID NO: 72) comprising an endogenous target site selected for modification. The encoded amino acid sequences are shown below each nucleotide sequence. (SEQ ID NOs: 73, 75, 77). FIG. 6A depicts single nucleotide (in this example C, shown in bold and underlined) located next to an endonuclease cleavage site (shown by arrow) can be removed through NHEJ. FIG. 6B depicts the resulting polynucleotide of interest (SEQ ID NO:74) having a single base deleted, resulting in the creation of a new cleavage site (indicated by arrow) and translational frameshift. FIG. 6C: A single nucleotide (in this example T, shown in bold and underlined) located next to an endonuclease cleavage site can be inserted through NHEJ without the use of a polynucleotide modification (repair) template, resulting in a single nucleotide edit of the polynucleotide of interest (SEQ ID NO:76). PAM sequences are highlighted in grey.

FIG. 7. Top: Agrobacterium vector for stable integration of the UBI:Cas9 into the maize genome. Bottom: Agrobacterium vector for stable integration of the MDH:Cas9 into the maize genome. MDH is a temperature regulated promoter, regulating expression of the Cas9. These vectors also contain visible marker gene (END2:AmCYAN), which was used for selection of stably transformed callus sectors. Sequence of the Red Fluorescent Protein (DsRED) contained duplicated in a direct orientation 369 bp fragments separated by a 343-bp spacer, which contained sequences for recognition and targeting by two gRNAs and LIG3:4 meganuclease. H2B refers to the histone H2B gene promoter.

SEQUENCES

TABLE 1 Summary of Nucleic Acid and Protein SEQ ID Numbers Nucleic acid Protein Description SEQ ID NO. SEQ ID NO. Cas9 coding sequence 1 potato ST-LS1 intron 2 SV40 NLS 3 VirD2 NLS 4 Maize optimized Cas9 expression cassette 5 Lig-CR3 guide RNA expression vector 6 Maize genomic target site MS26Cas-1 plus 7 PAM sequence Maize genomic target site MS26Cas-2 plus 8 PAM sequence Maize genomic target site MS26Cas-3 plus 9 PAM sequence Maize genomic target site LIGCas-1 plus 10 PAM sequence Maize genomic target site LIGCas-2 plus 11 PAM sequence Maize genomic target site LIGCas-3 plus 12 PAM sequence Maize genomic target site MS45Cas-1 plus 13 PAM sequence Maize genomic target site MS45Cas-2 plus 14 PAM sequence Maize genomic target site MS45Cas-3 plus 15 PAM sequence Maize genomic target site ALSCas-1 plus 16 PAM sequence Maize genomic target site ALSCas-2 plus 17 PAM sequence Maize genomic target site ALSCas-3 plus 18 PAM sequence Primer sequences 19-38 ALS1-DNA sequence 39 ALS2-DNA sequence 40 full length Zm-ALS2 protein 41 Maize genomic target site ALSCas-4 plus 42 PAM sequence 794 bp polynucleotide modification template 43 127 bp polynucleotide modification template, 44 oligo1 127 bp polynucleotide modification template, 45 oligo2 Agrobacterium vector containing maize 46 codon optimized Cas9 and maize UBI promoter Agrobacterium vector containing maize 47 codon optimized Cas9 and maize MDH promoter Sequences shown in FIG. 1 48-58 Sequences shown in FIG. 2 59, 61, 63 60, 62, 64 Sequences shown in FIG. 4A-4C 65, 67, 69 66, 68 Sequences shown in FIG. 5A-5B 70-71 Sequences shown in FIG. 6A-6C 72, 74, 76 73, 75, 77 IN2 promoter 78 ALSCas7 target site 79 ALSCas7-1 target site which is the modified 80 ALSCas7 target site maize off target site 81

DETAILED DESCRIPTION

Compositions and methods are provided for restoring function to a non-functional gene product in the genome of a cell. The methods and compositions employ a guide RNA/Cas endonuclease system to restore function to a non-functional gene product and to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. The present disclosure also describes methods for modifying a nucleotide sequence in the genome of a cell using a restored functional selectable marker, as well as methods for editing a nucleotide sequence in the genome a cell without introducing a polynucleotide modification template into said cell. Compositions and methods are also provided for DNA free delivery of Cas9 endonucleases, sgRNAs and guide RNA/Cas complexes by introducing the components of the complex (guide RNA and Cas endonucleases) via mRNA molecules (guide RNA, Cas endonuclease) or protein molecules (Cas endonuclease) or by directly introducing the nucleotide-protein complex into the cell.

CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats) (also known as SPIDRs—SPacer Interspersed Direct Repeats) constitute a family of DNA loci. CRISPR loci consist of short and highly conserved DNA repeats (typically 24 to 40 bp, repeated from 1 to 140 times—also referred to as CRISPR-repeats) which are partially palindromic. The repeated sequences (usually specific to a species) are interspaced by variable sequences of constant length (typically 20 to 58 by depending on the CRISPR locus (WO2007/025097, published Mar. 1, 2007). Bacteria and archaea have evolved adaptive immune defenses termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids (WO2007/025097, published Mar. 1, 2007). Multiple CRISPR-Cas systems have been described including Class 1 systems, with multisubunit effector complexes, and Class 2 systems, with single protein effectors (such as but not limiting to Cas9, Cpf1, C2c1, C2c2, C2c3). (Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15, WO 2013/176772 A1 published on Nov. 23, 2013 and incorporated by its entirety by reference herein).

The type II CRISPR/Cas system from bacteria employs a crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA) to guide the Cas endonuclease to its DNA target. The crRNA contains a spacer region complementary to one strand of the double strand DNA target and a region that base pairs with the tracrRNA (trans-activating CRISPR RNA) forming a RNA duplex that directs the Cas endonuclease to cleave the DNA target. Spacers are acquired through a not fully understood process involving Cas1 and Cas2 proteins. All type II CRISPR-Cas loci contain cas1 and cas2 genes in addition to the cas9 gene (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Cas gene includes a gene that is generally coupled, associated or close to, or in the vicinity of flanking CRISPR loci. The terms “Cas gene”, “CRISPR-associated (Cas) gene” are used interchangeably herein. A comprehensive review of the Cas protein family is presented in Haft et al. (2005) Computational Biology, PLoS Comput Biol 1(6): e60. doi:10.1371/journal.pcbi.0010060. As described therein, 41 CRISPR-associated (Cas) gene families are described, in addition to the four previously known gene families. It shows that CRISPR systems belong to different classes, with different repeat patterns, sets of genes, and species ranges. The number of Cas genes at a given CRISPR locus can vary between species (Haft et al., 2005, Computational Biology, PLoS Comput Biol 1(6): e60. doi:10.1371/journal.pcbi.0010060; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; WO 2013/176772 A1 published on Nov. 23, 2013 and incorporated by its entirety by reference herein).

The term “Cas endonuclease” herein refers to a protein encoded by a Cas (CRISPR-associated) gene. A Cas endonuclease, when in complex with a suitable polynucleotide component, is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific DNA target sequence. A Cas endonuclease described herein comprises one or more nuclease domains. Cas endonucleases of the disclosure include those having a HNH or HNH-like nuclease domain and/or a RuvC or RuvC-like nuclease domain (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). A Cas includes a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, Cas10, or complexes of these.

As used herein, the terms “guide polynucleotide/Cas endonuclease complex”, “guide polynucleotide/Cas endonuclease system”, “guide polynucleotide/Cas complex”, “guide polynucleotide/Cas system”, “guided Cas system”, “PGEN” are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease protein that are capable of forming a polynucleotide-protein complex, wherein said guide polynucleotide/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. A guide polynucleotide/Cas endonuclease complex herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any of the four known CRISPR systems (Horvath and Barrangou, Science 327:167-170) such as a type I, II, or III CRISPR system. A Cas endonuclease unwinds the DNA duplex at the target sequence and optionally cleaves at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, a crRNA or guide RNA) that is in complex with the Cas protein. Such recognition and cutting of a target sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3′ end of the DNA target sequence. Alternatively, a Cas protein herein may lack DNA cleavage or nicking activity, but can still specifically bind to a DNA target sequence when complexed with a suitable RNA component. (See also U.S. Patent Application US 2015-0082478 A1, published on Mar. 19, 2015 and US 2015-0059010 A1, published on Feb. 26, 2015, both are hereby incorporated in its entirety by reference).

A guide polynucleotide/Cas endonuclease complex can cleave one or both strands of a DNA target sequence. A guide polynucleotide/Cas endonuclease complex that can cleave both strands of a DNA target sequence typically comprises a Cas protein that has all of its endonuclease domains in a functional state (e.g., wild type endonuclease domains or variants thereof retaining some or all activity in each endonuclease domain). Thus, a wild type Cas protein (e.g., a Cas9 protein disclosed herein), or a variant thereof retaining some or all activity in each endonuclease domain of the Cas protein, is a suitable example of a Cas endonuclease that can cleave both strands of a DNA target sequence. A Cas9 protein comprising functional RuvC and HNH nuclease domains is an example of a Cas protein that can cleave both strands of a DNA target sequence. A guide polynucleotide/Cas endonuclease complex that can cleave one strand of a DNA target sequence can be characterized herein as having nickase activity (e.g., partial cleaving capability). A Cas nickase typically comprises one functional endonuclease domain that allows the Cas to cleave only one strand (i.e., make a nick) of a DNA target sequence. For example, a Cas9 nickase may comprise (i) a mutant, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., wild type HNH domain). As another example, a Cas9 nickase may comprise (i) a functional RuvC domain (e.g., wild type RuvC domain) and (ii) a mutant, dysfunctional HNH domain. Non-limiting examples of Cas9 nickases suitable for use herein are disclosed by Gasiunas et al. (Proc. Natl. Acad. Sci. U.S.A. 109:E2579-E2586), Jinek et al. (Science 337:816-821), Sapranauskas et al. (Nucleic Acids Res. 39:9275-9282) and in U.S. Patent Appl. Publ. No. 2014/0189896, which are incorporated herein by reference.

A pair of Cas9 nickases can be used to increase the specificity of DNA targeting. In general, this can be done by introducing two Cas9 nickases that, by virtue of being associated with RNA components with different guide sequences, target and nick nearby DNA sequences on opposite strands in the region for desired targeting. Such nearby cleavage of each DNA strand creates a double strand break (i.e., a DSB with single-stranded overhangs), which is then recognized as a substrate for non-homologous-end-joining, NHEJ (prone to imperfect repair leading to mutations) or homologous recombination, HR. Each nick in these embodiments can be at least about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 (or any integer between 5 and 100) bases apart from each other, for example. One or two Cas9 nickase proteins herein can be used in a Cas9 nickase pair. For example, a Cas9 nickase with a mutant RuvC domain, but functioning HNH domain (i.e., Cas9 HNH+/RuvC−), could be used (e.g., Streptococcus pyogenes Cas9 HNH+/RuvC−). Each Cas9 nickase (e.g., Cas9 HNH+/RuvC−) would be directed to specific DNA sites nearby each other (up to 100 base pairs apart) by using suitable RNA components herein with guide RNA sequences targeting each nickase to each specific DNA site.

A Cas protein can be part of a fusion protein comprising one or more heterologous protein domains (e.g., 1, 2, 3, or more domains in addition to the Cas protein). Such a fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains, such as between Cas and a first heterologous domain. Examples of protein domains that may be fused to a Cas protein herein include, without limitation, epitope tags (e.g., histidine [His], V5, FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]), reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], beta-galactosidase, beta-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]), and domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity (e.g., VP16 or VP64), transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. A Cas protein can also be in fusion with a protein that binds DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16.

A Cas protein herein can be from any of the following genera: Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Haloarcula, Methanobacteriumn, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Themioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphvromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Streptococcus, Treponema, Francisella, or Thermotoga. Alternatively, a Cas protein herein can be encoded, for example, by any of SEQ ID NOs:462-465, 467-472, 474-477, 479-487, 489-492, 494-497, 499-503, 505-508, 510-516, or 517-521 as disclosed in U.S. Appl. Publ. No. 2010/0093617, which is incorporated herein by reference.

A guide polynucleotide/Cas endonuclease complex in certain embodiments can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence. Such a complex may comprise a Cas protein in which all of its nuclease domains are mutant, dysfunctional. For example, a Cas9 protein herein that can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence, may comprise both a mutant, dysfunctional RuvC domain and a mutant, dysfunctional HNH domain. A Cas protein herein that binds, but does not cleave, a target DNA sequence can be used to modulate gene expression, for example, in which case the Cas protein could be fused with a transcription factor (or portion thereof) (e.g., a repressor or activator, such as any of those disclosed herein).

The Cas endonuclease gene can be a Type II Cas9 endonuclease, such as but not limited to, Cas9 genes listed in SEQ ID NOs: 462, 474, 489, 494, 499, 505, and 518 of WO2007/025097 published Mar. 1, 2007, and incorporated herein by reference. In another embodiment, the Cas endonuclease gene is a plant, maize or soybean optimized Cas9 endonuclease gene. The Cas endonuclease gene herein can be a plant or microbial codon optimized Cas9 endonuclease gene. The Cas endonuclease gene can be operably linked to a SV40 nuclear targeting signal upstream of the Cas codon region and a bipartite VirD2 nuclear localization signal (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442-6) downstream of the Cas codon region.

“Cas9” (formerly referred to as Cas5, Csn1, or Csx12) herein refers to a Cas endonuclease of a type II CRISPR system that forms a complex with a crNucleotide and a tracrNucleotide, or with a single guide polynucleotide, for specifically recognizing and cleaving all or part of a DNA target sequence. A Cas9 protein comprises a RuvC nuclease domain and an HNH (H-N-H) nuclease domain, each of which can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick). In general, the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al, Cell 157:1262-1278). A type II CRISPR system includes a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one polynucleotide component. For example, a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex with a single guide RNA.

The amino acid sequence of a Cas9 protein described herein, as well as certain other Cas proteins herein, may be derived from a Streptococcus (e.g., S. pyogenes, S. pneumoniae, S. thermophilus, S. agalactiae, S. parasanguinis, S. oralis, S. salivarius, S. macacae, S. dysgalactiae, S. anginosus, S. constellatus, S. pseudoporcinus, S. mutans), Listeria (e.g., L. innocua), Spiroplasma (e.g., S. apis, S. syrphidicola), Peptostreptococcaceae, Atopobium, Porphyromonas (e.g., P. catoniae), Prevotella (e.g., P. intermedia), Veillonella, Treponema (e.g., T. socranskii, T. denticola), Capnocytophaga, Finegoldia (e.g., F. magna), Coriobacteriaceae (e.g., C. bacterium), Olsenella (e.g., O. profusa), Haemophilus (e.g., H. sputorum, H. pittmaniae), Pasteurella (e.g., P. bettyae), Olivibacter (e.g., O. sitiensis), Epilithonimonas (e.g., E. tenax), Mesonia (e.g., M. mobilis), Lactobacillus (e.g., L. plantarum), Bacillus (e.g., B. cereus), Aquimarina (e.g., A. muellen), Chryseobacterium (e.g., C. palustre), Bacteroides (e.g., B. graminisolvens), Neisseria (e.g., N. meningitidis), Francisella (e.g., F. novicida), or Flavobacterium (e.g., F. frigidarium, F. soli) species, for example. As another example, a Cas9 protein can be any of the Cas9 proteins disclosed in Chylinski et al. (RNA Biology 10:726-737 and U.S. patent application 62/162,377, filed May 15, 2015), which are incorporated herein by reference.

Accordingly, the sequence of a Cas9 protein herein can comprise, for example, any of the Cas9 amino acid sequences disclosed in GenBank Accession Nos. G3ECR1 (S. thermophilus), WP_026709422, WP_027202655, WP_027318179, WP_027347504, WP_027376815, WP_027414302, WP_027821588, WP_027886314, WP_027963583, WP_028123848, WP_028298935, Q03JI6 (S. thermophilus), EGP66723, EGS38969, EGV05092, EHI65578 (S. pseudoporcinus), EIC75614 (S. oralis), EID22027 (S. constellatus), EIJ69711, EJP22331 (S. oralis), EJP26004 (S. anginosus), EJP30321, EPZ44001 (S. pyogenes), EPZ46028 (S. pyogenes), EQL78043 (S. pyogenes), EQL78548 (S. pyogenes), ERL10511, ERL12345, ERL19088 (S. pyogenes), ESA57807 (S. pyogenes), ESA59254 (S. pyogenes), ESU85303 (S. pyogenes), ETS96804, UC75522, EGR87316 (S. dysgalactiae), EGS33732, EGV01468 (S. oralis), EHJ52063 (S. macacae), EID26207 (S. oralis), EID33364, EIG27013 (S. parasanguinis), EJF37476, EJO19166 (Streptococcus sp. BS35b), EJU16049, EJU32481, YP_006298249, ERF61304, ERK04546, ETJ95568 (S. agalactiae), TS89875, ETS90967 (Streptococcus sp. SR4), ETS92439, EUB27844 (Streptococcus sp. BS21), AFJ08616, EUC82735 (Streptococcus sp. CM6), EWC92088, EWC94390, EJP25691, YP_008027038, YP_008868573, AGM26527, AHK22391, AHB36273, Q927P4, G3ECR1, or Q99ZW2 (S. pyogenes), which are incorporated by reference. A variant of any of these Cas9 protein sequences may be used, but should have specific binding activity, and optionally endonucleolytic activity, toward DNA when associated with an RNA component herein. Such a variant may comprise an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the reference Cas9.

Alternatively, a Cas9 protein herein can be encoded by any of SEQ ID NOs:462 (S. thermophilus), 474 (S. thermophilus), 489 (S. agalactiae), 494 (S. agalactiae), 499 (S. mutans), 505 (S. pyogenes), or 518 (S. pyogenes) as disclosed in U.S. Appl. Publ. No. 2010/0093617 (incorporated herein by reference), for example. Alternatively still, a Cas9 protein may comprise an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing amino acid sequences, for example. Such a variant Cas9 protein should have specific binding activity, and optionally cleavage or nicking activity, toward DNA when associated with an RNA component herein.

A Cas protein herein such as a Cas9 can comprise a heterologous nuclear localization sequence (NLS). A heterologous NLS amino acid sequence herein may be of sufficient strength to drive accumulation of a Cas protein in a detectable amount in the nucleus of a yeast cell herein, for example. An NLS may comprise one (monopartite) or more (e.g., bipartite) short sequences (e.g., 2 to 20 residues) of basic, positively charged residues (e.g., lysine and/or arginine), and can be located anywhere in a Cas amino acid sequence but such that it is exposed on the protein surface. An NLS may be operably linked to the N-terminus or C-terminus of a Cas protein herein, for example. Two or more NLS sequences can be linked to a Cas protein, for example, such as on both the N- and C-termini of a Cas protein. Non-limiting examples of suitable NLS sequences herein include those disclosed in U.S. Pat. Nos. 6,660,830 and 7,309,576 (e.g., Table 1 therein), which are both incorporated herein by reference.

The Cas endonuclease can comprise a modified form of the Cas9 polypeptide. The modified form of the Cas9 polypeptide can include an amino acid change (e.g., deletion, insertion, or substitution) that reduces the naturally-occurring nuclease activity of the Cas9 protein. For example, in some instances, the modified form of the Cas9 protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 polypeptide (US patent application US20140068797 A1, published on Mar. 6, 2014). In some cases, the modified form of the Cas9 polypeptide has no substantial nuclease activity and is referred to as catalytically “inactivated Cas9” or “deactivated cas9 (dCas9).” Catalytically inactivated Cas9 variants include Cas9 variants that contain mutations in the HNH and RuvC nuclease domains. These catalytically inactivated Cas9 variants are capable of interacting with sgRNA and binding to the target site in vivo but cannot cleave either strand of the target DNA.

A catalytically inactive Cas9 can be fused to a heterologous sequence (US patent application US20140068797 A1, published on Mar. 6, 2014). Suitable fusion partners include, but are not limited to, a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide (e.g., a histone or other DNA-binding protein) associated with the target DNA. Additional suitable fusion partners include, but are not limited to, a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. Further suitable fusion partners include, but are not limited to, a polypeptide that directly provides for increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription regulator, etc.). A catalytically inactive Cas9 can also be fused to a FokI nuclease to generate double strand breaks (Guilinger et al. Nature biotechnology, volume 32, number 6, June 2014).

The terms “functional fragment”, “fragment that is functionally equivalent” and “functionally equivalent fragment” of a Cas endonuclease are used interchangeably herein, and refer to a portion or subsequence of a Cas endonuclease sequence in which the ability to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break in) the target site is retained.

The terms “functional variant”, “Variant that is functionally equivalent” and “functionally equivalent variant” of a Cas endonuclease are used interchangeably herein, and refer to a variant of a Cas endonuclease in which the ability to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break in) the target site is retained. Fragments and variants can be obtained via methods such as site-directed mutagenesis and synthetic construction.

The Cas endonuclease gene includes a plant codon optimized Streptococcus pyogenes Cas9 gene that can recognize any genomic sequence of the form N(12-30)NGG can in principle be targeted or a Cas9 endonuclease originated from an organism selected from the group consisting of Brevibacillus laterosporus, Lactobacillus reuteri Mlc3, Lactobacillus rossiae DSM 15814, Pediococcus pentosaceus SL4, Lactobacillus nodensis JCM 14932, Sulfurospirillum sp. SCADC, Bifidobacterium thermophilum DSM 20210, Loktanella vestfoldensis, Sphingomonas sanxanigenens NX02, Epilithonimonas tenax DSM 16811, Sporocytophaga myxococcoides and Psychroflexus torquis ATCC 700755, wherein said Cas9 endonuclease can form a guide RNA/Cas endonuclease complex capable of recognizing, binding to, and optionally nicking or cleaving all or part of a DNA target sequence. Other Cas endonuclease systems have been described in U.S. patent applications 62/162,377 filed May 15, 2015 and 62/162,353 filed May 15, 2015, both applications incorporated herein by reference.

Cas9 endonucleases can be used for targeted genome editing (via simplex and multiplex double-strand breaks and nicks) and targeted genome regulation (via tethering of epigenetic effector domains to either the Cas9 or sgRNA. Cas9 might also be engineered to function as an RNA-guided recombinase, and via RNA tethers could serve as a scaffold for the assembly of multiprotein and nucleic acid complexes (Mali et al. 2013 Nature Methods Vol. 10: 957-963.).

As used herein, the term “guide polynucleotide”, relates to a polynucleotide sequence that can form a complex with a Cas endonuclease and enables the Cas endonuclease to recognize, bind to, and optionally cleave a DNA target site. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be a RNA sequence (referred to as guide RNA, gRNA), a DNA sequence, or a combination thereof (a RNA-DNA combination sequence). Optionally, the guide polynucleotide can comprise at least one nucleotide, phosphodiester bond or linkage modification such as, but not limited, to Locked Nucleic Acid (LNA), 5-methyl dC, 2,6-Diaminopurine, 2′-Fluoro A, 2′-Fluoro U, 2′-O-Methyl RNA, phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule, or 5′ to 3′ covalent linkage resulting in circularization. A guide polynucleotide that solely comprises ribonucleic acids is also referred to as a “guide RNA” or “gRNA” (See also U.S. Patent Application US 2015-0082478 A1, published on Mar. 19, 2015 and US 2015-0059010 A1, published on Feb. 26, 2015, both are hereby incorporated in its entirety by reference).

The guide polynucleotide can be a double molecule (also referred to as duplex guide polynucleotide) comprising a crNucleotide sequence and a tracrNucleotide sequence. The crNucleotide includes a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA and a second nucleotide sequence (also referred to as a tracr mate sequence) that is part of a Cas endonuclease recognition (CER) domain. The tracr mate sequence can hybridized to a tracrNucleotide along a region of complementarity and together form the Cas endonuclease recognition domain or CER domain. The CER domain is capable of interacting with a Cas endonuclease polypeptide. The crNucleotide and the tracrNucleotide of the duplex guide polynucleotide can be RNA, DNA, and/or RNA-DNA-combination sequences. In some embodiments, the crNucleotide molecule of the duplex guide polynucleotide is referred to as “crDNA” (when composed of a contiguous stretch of DNA nucleotides) or “crRNA” (when composed of a contiguous stretch of RNA nucleotides), or “crDNA-RNA” (when composed of a combination of DNA and RNA nucleotides). The crNucleotide can comprise a fragment of the crRNA naturally occurring in Bacteria and Archaea. The size of the fragment of the crRNA naturally occurring in Bacteria and Archaea that can be present in a crNucleotide disclosed herein can range from, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments the tracrNucleotide is referred to as “tracrRNA” (when composed of a contiguous stretch of RNA nucleotides) or “tracrDNA” (when composed of a contiguous stretch of DNA nucleotides) or “tracrDNA-RNA” (when composed of a combination of DNA and RNA nucleotides. In one embodiment, the RNA that guides the RNA/Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA.

The tracrRNA (trans-activating CRISPR RNA) contains, in the 5′-to-3′ direction, (i) a sequence that anneals with the repeat region of CRISPR type II crRNA and (ii) a stem loop-containing portion (Deltcheva et al., Nature 471:602-607). The duplex guide polynucleotide can form a complex with a Cas endonuclease, wherein said guide polynucleotide/Cas endonuclease complex (also referred to as a guide polynucleotide/Cas endonuclease system) can direct the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) into the target site. (See also U.S. Patent Application US 2015-0082478 A1, published on Mar. 19, 2015 and US 2015-0059010 A1, published on Feb. 26, 2015, both are hereby incorporated in its entirety by reference.)

The guide polynucleotide can also be a single molecule (also referred to as single guide polynucleotide) comprising a crNucleotide sequence linked to a tracrNucleotide sequence. The single guide polynucleotide comprises a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA and a Cas endonuclease recognition domain (CER domain), that interacts with a Cas endonuclease polypeptide. By “domain” it is meant a contiguous stretch of nucleotides that can be RNA, DNA, and/or RNA-DNA-combination sequence. The VT domain and/or the CER domain of a single guide polynucleotide can comprise a RNA sequence, a DNA sequence, or a RNA-DNA-combination sequence. The single guide polynucleotide being comprised of sequences from the crNucleotide and the tracrNucleotide may be referred to as “single guide RNA” (when composed of a contiguous stretch of RNA nucleotides) or “single guide DNA” (when composed of a contiguous stretch of DNA nucleotides) or “single guide RNA-DNA” (when composed of a combination of RNA and DNA nucleotides). The single guide polynucleotide can form a complex with a Cas endonuclease, wherein said guide polynucleotide/Cas endonuclease complex (also referred to as a guide polynucleotide/Cas endonuclease system) can direct the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the target site. (See also U.S. Patent Application US 2015-0082478 A1, published on Mar. 19, 2015 and US 2015-0059010 A1, published on Feb. 26, 2015, both are hereby incorporated in its entirety by reference.)

The term “variable targeting domain” or “VT domain” is used interchangeably herein and includes a nucleotide sequence that can hybridize (is complementary) to one strand (nucleotide sequence) of a double strand DNA target site. The % complementation between the first nucleotide sequence domain (VT domain) and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The variable targeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the variable targeting domain comprises a contiguous stretch of 12 to 30 nucleotides. The variable targeting domain can be composed of a DNA sequence, a RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

The term “Cas endonuclease recognition domain” or “CER domain” (of a guide polynucleotide) is used interchangeably herein and includes a nucleotide sequence that interacts with a Cas endonuclease polypeptide. A CER domain comprises a tracrNucleotide mate sequence followed by a tracrNucleotide sequence. The CER domain can be composed of a DNA sequence, a RNA sequence, a modified DNA sequence, a modified RNA sequence (see for example US 2015-0059010 A1, published on Feb. 26, 2015, incorporated in its entirety by reference herein), or any combination thereof.

The nucleotide sequence linking the crNucleotide and the tracrNucleotide of a single guide polynucleotide can comprise a RNA sequence, a DNA sequence, or a RNA-DNA combination sequence. In one embodiment, the nucleotide sequence linking the crNucleotide and the tracrNucleotide of a single guide polynucleotide can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length. In another embodiment, the nucleotide sequence linking the crNucleotide and the tracrNucleotide of a single guide polynucleotide can comprise a tetraloop sequence, such as, but not limiting to a GAAA tetraloop sequence.

Nucleotide sequence modification of the guide polynucleotide, VT domain and/or CER domain can be selected from, but not limited to, the group consisting of a 5′ cap, a 3′ polyadenylated tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide poly nucleotide to a subcellular location, a modification or sequence that provides for tracking, a modification or sequence that provides a binding site for proteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a 2,6-Diaminopurine nucleotide, a 2′-Fluoro A nucleotide, a 2′-Fluoro U nucleotide; a 2′-O-Methyl RNA nucleotide, a phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 molecule, a 5′ to 3′ covalent linkage, or any combination thereof. These modifications can result in at least one additional beneficial feature, wherein the additional beneficial feature is selected from the group of a modified or regulated stability, a subcellular targeting, tracking, a fluorescent label, a binding site for a protein or protein complex, modified binding affinity to complementary target sequence, modified resistance to cellular degradation, and increased cellular permeability.

The terms “functional fragment”, “fragment that is functionally equivalent” and “functionally equivalent fragment” of a guide RNA, crRNA or tracrRNA are used interchangeably herein, and refer to a portion or subsequence of the guide RNA, crRNA or tracrRNA, respectively, in which the ability to function as a guide RNA, crRNA or tracrRNA, respectively, is retained.

The terms “functional variant”, “Variant that is functionally equivalent” and “functionally equivalent variant” of a guide RNA, crRNA or tracrRNA (respectively) are used interchangeably herein, and refer to a variant of the guide RNA, crRNA or tracrRNA, respectively, in which the ability to function as a guide RNA, crRNA or tracrRNA, respectively, is retained.

The terms “single guide RNA”, “gRNA” and “sgRNA” are used interchangeably herein and relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain (linked to a tracr mate sequence that hybridizes to a tracrRNA), fused to a tracrRNA (trans-activating CRISPR RNA). The single guide RNA can comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of the type II CRISPR/Cas system that can form a complex with a type II Cas endonuclease, wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site.

The terms “guide RNA/Cas endonuclease complex”, “guide RNA/Cas endonuclease system”, “guide RNA/Cas complex”, “guide RNA/Cas system”, “gRNA/Cas complex”, “gRNA/Cas system”, “RNA-guided endonuclease”, “RGEN” are used interchangeably herein and refer to at least one RNA component and at least one Cas endonuclease protein that are capable of forming a complex, wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. A guide RNA/Cas endonuclease complex herein can comprise Cas protein(s) and suitable RNA component(s) of any of the known CRISPR systems (Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular_Cell 60, 1-13; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; Horvath and Barrangou, Science 327:167-170) such as a type I, II, or III CRISPR system. A guide RNA/Cas endonuclease complex can comprise a Type II Cas9 endonuclease and at least one RNA component (e.g., a crRNA and tracrRNA, or a gRNA). (See also U.S. Patent Application US 2015-0082478 A1, published on Mar. 19, 2015 and US 2015-0059010 A1, published on Feb. 26, 2015, both are hereby incorporated in its entirety by reference).

The Cas endonuclease can be introduced into a cell (provided to a cell) by any method known in the art, for example, but not limited to transient introduction methods, transfection, microinjection, and/or topical application or indirectly via recombination constructs. Plant cells differ from human and animal cells in that plant cells contain a plant cell wall which may act as a barrier to the direct delivery of the Cas9 endonuclease into the plant cell. Recombinant DNA constructs encoding a Cas9 endonuclease have been successfully introduced into plant cells (Svitashev et al., Plant Physiology, 2015, Vol. 169, pp. 931-945) to allow for genome editing at a target site. One possible disadvantage of stably introducing recombinant DNA constructs in plant cells is that the continued presence of Cas9 endonucleases may increase off-target effects.

As described herein, direct delivery of the Cas endonuclease into plant cells can be achieved through particle mediated delivery. Based on the experiments described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, whisker mediated delivery, electroporation, particle bombardment, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering the Cas9 endonuclease in plant cells.

Direct delivery of the Cas endonuclease (also referred to as DNA free delivery off the Cas endonuclease) can be achieved by introducing the Cas protein, the mRNA encoding the Cas endonuclease, and/or the RNA guided endonuclease ribonucleotide-protein complex (RGEN) itself (as a ribonucleotide-protein complex), into a cell using any method known in the art. Direct delivery of the Cas endonuclease, either via mRNA encoding the Cas endonuclease or via a polypeptide molecule is also referred to herein as DNA free delivery of the Cas endonuclease, as no DNA molecule is involved in the production of the Cas endonuclease protein. Similarly, direct delivery of the guide RNA as an RNA molecule is also referred to herein as DNA free delivery of the guide RNA. Similarly, direct delivery of the guide RNA/endonuclease complex itself (RGEN) as a ribonucleotide-protein complex, is also referred to herein as DNA free delivery of the RGEN.

Directly introducing the Cas endonuclease as a protein, or as an mRNA molecule together with a gRNA, or as a RGEN ribonucleotide-protein itself, allows for genome editing at the target site followed by rapid degradation of the RGEN complex, and only a transient presence of the complex in the cell which leads to reduced off-target effects (as described in Example 12).

Direct delivery of these components can be accompanied by direct delivery (co-delivery) of other mRNAs that can promote the enrichment and/or visualization of cells receiving the RGEN components. For example, delivery of mRNAs encoding screenable visual markers such as fluorescence proteins (for example but not limited to Red, green, yellow, blue or combinations thereof) can also be used in lieu of, or coupled with, direct selection of a repaired disrupted, non-functional gene product.

Described herein are methods to restore the function of a non-functional gene product by restoring the nucleotide sequence of a disrupted gene such that the restored nucleotide sequence encodes the functional gene product.

A disrupted gene refers to a gene that has been modified (disrupted) such that its gene product loses its function (referred to as a non-functional gene product) or has a reduced function when compared to the product of the corresponding gene that does not have the disruption (also referred to as the undisrupted gene). For example, a gene encoding for a functional polypeptide or protein can be disrupted (modified) such that the translation product of the disrupted gene results in a polypeptide that has lost its function or has a reduced function.

A functional gene product includes a functional protein or polypeptide that has a biological or non-biological function.

A non-functional gene product includes reference to the gene product of a disrupted gene. The non-functional gene product includes polypeptides that have lost their function (absent function) or have a reduced function when compared to the gene product of the corresponding undisrupted gene.

In one embodiment of the disclosure, the method comprises a method for restoring function to a non-functional gene product in the genome of a cell, the method comprising introducing a guide RNA/Cas endonuclease complex into a cell comprising a disrupted gene in its genome, wherein said complex creates a double strand break, wherein said disrupted gene does not encode a functional gene product, wherein said disrupted gene is restored without the use of a polynucleotide modification template to a non-disrupted gene capable of encoding said functional gene product. The disrupted gene can comprise a base pair deletion of the 4^(th) nucleotide upstream (5′) of a PAM sequence when compared to its corresponding non-disrupted gene, wherein said base pair deletion creates an amino acid frameshift in the gene product of the disrupted gene thereby rendering the gene product of the disrupted gene non-functional. The base pair deletion can be the first, second or third nucleotide of a codon sequence. The restoration can be accomplished by Non-Homologous-End-Joining (NHEJ) resulting in the insertion of a single base at the double strand break site, or can be accomplished by the insertion of a single base at the double strand break site without the use of Homologous Recombination or Homology Directed Repair.

Coincident with the restoration of gene function by NHEJ (through for example delivery of RGEN components or the RGEN complex itself to a cell), modification of other targets can be accomplished by the simultaneous addition of other guide-polynucleotides. Such other targets (other than the target for restoration of gene function by NHEJ) can be any target in the genome including a transgenic locus. The approach of simultaneous delivery of two or more gRNAs when one gRNA targets and activates a selectable marker through NHEJ, (such as but not limiting to conferring herbicide tolerance) and the other gRNA(s) promote DSB(s) at target site(s) different than the selectable marker (or other disrupted gene design) and can facilitate either targeted mutagenesis, deletion, gene editing, or site-specific trait gene insertions can allow for completely transient targeted genome modifications as all other necessary components (Cas9, gRNAs) can be delivered in a form of protein and/or in vitro transcribed RNA molecules.

A disrupted gene includes reference to a marker gene (such as, but not limited to, a phenotypic marker gene and a selectable marker gene) that has been modified (disrupted) such that its gene product loses its function (for example, in case of a herbicide disrupted selectable marker gene, the disrupted gene does not confer herbicide resistance anymore).

A selectable marker and screenable marker are used interchangeably herein and includes reference to a DNA segment (such as a selectable marker gene) that allows one to identify, or select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like. A selectable marker further includes a gene that when modified or knocked-out generates a property in a cell that allows one to identify, or select for (or against) a cell that contains said property.

In one aspect, the selectable marker allows for the selection of cells by applying selection schemes, in which for example, a selective agent (such as, but not limited to an antibiotic or herbicide) is used to inhibit or kill cells or tissues that do not comprise the selectable marker, and the cells or tissues that comprise the selectable marker continue to grow due to expression of the selectable marker gene.

In one aspect, the selectable marker allows for the visual selection of cells by applying selection schemes, in which for example, a visible marker (such as a fluorescent molecule) is used to select cells that comprise the visible marker.

Selectable marker genes include, but are not limited to, chlorosulfuron resistance genes, phosphomannose isomerase genes (PMI), bialaphos resistance genes (BAR), phosphinothricin acetyltransferase (PAT) genes, hygromycin resistance genes (NPTII), glyphosate resistance genes, DNA segments that comprise restriction enzyme sites; DNA segments that encode products which provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT)); DNA segments that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA segments that encode products which can be readily identified (referred to as visible maker genes. Visible marker genes include reference to fluorescent markers genes, such as red fluorescent marker genes, blue fluorescent marker genes, green fluorescent marker genes, yellow fluorescent marker genes), genes encoding DsRED, RFP, red fluorescent protein, CFP, GFP, green fluorescent protein) and genes encoding phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cell surface proteins. Selectable marker genes further include the generation of new primer sites for PCR (e.g., the juxtaposition of two DNA sequence not previously juxtaposed), the inclusion of DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; and, the inclusion of a DNA sequences required for a specific modification (e.g., methylation) that allows its identification.

Additional selectable markers include genes that confer resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See for example, Yarranton, (1992) Curr Opin Biotech 3:506-11; Christopherson et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-8; Yao et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol Microbiol 6:2419-22; Hu et al., (1987) Cell 48:555-66; Brown et al., (1987) Cell 49:603-12; Figge et al., (1988) Cell 52:713-22; Deuschle et al., (1989) Proc. Natl. Acad. Sci. USA 86:5400-4; Fuerst et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-53; Deuschle et al., (1990) Science 248:480-3; Gossen, (1993) Ph.D. Thesis, University of Heidelberg; Reines et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-21; Labow et al., (1990) Mol Cell Biol 10:3343-56; Zambretti et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-6; Baim et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-6; Wyborski et al., (1991) Nucleic Acids Res 19:4647-53; Hillen and Wissman, (1989) Topics Mol Struc Biol 10:143-62; Degenkolb et al., (1991) Antimicrob Agents Chemother 35:1591-5; Kleinschnidt et al., (1988) Biochemistry 27:1094-104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-51; Oliva et al., (1992) Antimicrob Agents Chemother 36:913-9; Hlavka et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al., (1988) Nature 334:721-4.

Phenotypic marker genes include genes encoding a screenable or selectable marker that includes visual markers, whether it is a positive or negative selectable marker. Any phenotypic marker can be used.

As described herein, a phenotypic and selectable marker gene can be modified to be introduced into plant cells as a disrupted gene encoding a non-functional gene product and used as targets by double strand break inducing endonucleases for restoration back to the non-disrupted gene encoding a functional gene product, by guide RNA introduction and DNA repair.

Phenotypic or selectable marker genes to be disrupted can be marker genes that were previously introduced into the cell and are stably incorporated into the genome of the cell. Such pre-integrated selectable marker genes can also be complemented with other genes, for example, cell developmental enhancing genes (ZmODP2 and ZmWUS, see for example PCT/US16/49144, filed Aug. 26, 2016 and PCT/US16/49128 filed Aug. 26, 2016, incorporated herein by reference).

As described herein, the phenotypic and selectable marker genes can be modified to be introduced into plant cells as disrupted genes (inactive forms) and used as targets by double strand break inducing endonucleases for restoration (re-activation) by guide RNA introduction and repair.

Described herein are expression markers genes (such as but not limiting to ALS, EPSPS, BAR) that confers resistance to a compound or allows the endogenous or previously integrated marker gene or its gene product to be used as a marker, that can be modified into inactive forms and then used as targets for re-activation by guide RNA introduction and repair as described herein (see for Example 3-4).

In one embodiment of the disclosure, the method comprises a method for modifying a nucleotide sequence in the genome of a cell, the method comprising: introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by or based upon said functional selectable marker protein. The selection can include providing a chemical to the cell (such as a plant cell, or plants derived from said plant cell) expressing said functional selectable marker protein, wherein said functional selectable marker protein makes the cell (such as a plant cell, or plants derived from said plant cell) resistant to said chemical. The chemical can be provided during any developmental stage of the cell (in case of a plant cell during any stage of plant cell or plant development) to select for the cells or organisms comprising the desired modification in their genome. The selection also includes screening of cells (such as a plant cell, or plants derived from said plant cell) for the presence of a visual marker that results from the expression of said functional selectable marker protein.

For example, as described herein, the method can be a method for modifying a nucleotide sequence in the genome of a cell, the method comprising: introducing into at least one cell comprising a target site and a disrupted resistant-ALS marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted resistant-ALS marker gene, wherein said disrupted resistance-ALS marker gene is restored without the use of a polynucleotide modification template to a non-disrupted resistant ALS-marker gene capable of encoding a functional resistance-ALS protein that confers resistance to chlorsulfuron, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by or based upon said resistance-ALS protein that confers resistance to chlorsulfuron, The selection step can include providing chlorsulfuron to the plant cell or plants derived from said plant cell at any stage during plant cell or plant development to select for the plant cell or plants comprising the desired modification the their genome.

The introducing and selection step does not comprise the introduction of a selectable marker gene, such as a recombinant DNA construct comprising a selectable marker gene. The disrupted selectable marker gene can be any disrupted marker gene including a disrupted visible marker gene. The modification in the targeted nucleotide sequence can be selected from the group consisting of an insertion of at least one nucleotide, a deletion of at least one nucleotide, or a substitution of at least one nucleotide in said target site. The method can further comprise introducing a polynucleotide modification template into said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence, or a donor DNA wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site.

Any guided endonuclease can be used in the methods disclosed herein. Such endonucleases include, but are not limited to Cas and Cpf1 endonucleases. Many endonucleases have been described to date that can recognize specific PAM sequences (see for example—U.S. patent applications 62/162,377 filed May 15, 2015 and 62/162,353 filed May 15, 2015 and Zetsche B et al. 2015. Cell 163, 1013) and cleave the target DNA at a specific positions. It is understood that based on the methods and embodiments described herein utilizing a guided Cas system one can now tailor these methods such that they can utilize any guided endonuclease system. For example, one can envision adapting the method for restoring function to a non-functional gene product in the genome of a cell described herein to a method comprising introducing a guided Cpf1 endonuclease complex instead of a guided Cas endonuclease complex to restore a disrupted gene and creating a functional gene product. Other guided endonucleases and nucleotide-protein complexes that find use in the methods disclosed herein include those described in WO 2013/088446.

Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, and include restriction endonucleases that cleave DNA at specific sites without damaging the bases. Restriction endonucleases include Type I, Type II, Type III, and Type IV endonucleases, which further include subtypes. In the Type I and Type III systems, both the methylase and restriction activities are contained in a single complex. Endonucleases also include meganucleases, also known as homing endonucleases (HEases), which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18 bp or more (patent application PCT/US12/30061, filed on Mar. 22, 2012). Meganucleases have been classified into four families based on conserved sequence motifs, the families are the LAGLIDADG, GIY-YIG, H-N-H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganuclease is similar to the convention for other restriction endonuclease. Meganucleases are also characterized by prefix F-, I-, or PI—for enzymes encoded by free-standing ORFs, introns, and inteins, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. This cleaving activity can be used to produce a double-strand break. For reviews of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7. In some examples the recombinase is from the Integrase or Resolvase families.

TAL effector nucleases are a new class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism. (Miller et al. (2011) Nature Biotechnology 29:143-148). Zinc finger nucleases (ZFNs) are engineered double-strand break inducing agents comprised of a zinc finger DNA binding domain and a double-strand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprising two, three, or four zinc fingers, for example having a C2H2 structure, however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example nuclease domain from a Type IIs endonuclease such as FokI. Additional functionalities can be fused to the zinc-finger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases. In some examples, dimerization of nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3 finger domain recognized a sequence of 9 contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind an 18 nucleotide recognition sequence.

DNA double strand break (DSB) technologies (ZFNs, TALENs and CRISPR-Cas) have wide-ranging applications in academic research, gene therapy, and animal and plant breeding programs. These technologies have been successfully used to introduce genome modifications in multiple plant species, including major crops such as maize, wheat, soybean and rice. Plant genome editing is limited by current transformation and gene modification methods, efficiency of DNA delivery, and low frequencies of plant regeneration. In contrast to human and animal systems, the presence of a thick wall surrounding every plant cell fundamentally impacts plant transformation and plant gene modification protocols. This cell wall makes it impossible to use transfection or electroporation, which are broadly used for nucleic acid and/or protein delivery in mammalian genome editing experiments. For this reason, plant transformation and plant genome modification primarily relies on Agrobacterium-mediated and biolistic delivery (ballistic delivery) of guide RNA/Cas endonuclease reagents on DNA vectors. As a result, gRNA and Cas9 expression cassettes frequently integrate into the genome and potentially lead to gene disruption, plant mosaicism, and potential off-site cutting. Although these undesired secondary changes can be segregated away by several rounds of backcrossing to the wild type parent plant, this process can be time consuming especially for crops with complex polyploid genomes and long breeding cycles such as, but not limited to, soybean and wheat. As described herein, delivery of Cas endonuclease and gRNAs in the form of RGEN complexes into plant cells can mitigate many of these side effects (Example 11-12). An unexpected high frequency of NHEJ-mediated mutagenesis facilitated by delivery of RGEN complexes in plants is described in Example 10. Given this high frequency of mutagenesis using a RGEN complex, DNA-free and selectable marker-free gene modification may become a practical approach to generate gene knock-outs. This DNA- and selectable marker-free approach might be less practical for gene editing and gene insertion (as compared to gene mutations by NHEJ) applications due to the low frequency of the HDR pathway in somatic plant cells. Moreover, DNA molecules often integrate into the targeted DSB sites, decreasing the efficiency of gene editing, and especially, gene insertion. It has been demonstrated that DNA vectors encoding for genes delivered into the plant cell (for example, Cas9, gRNA, selectable marker genes and trait gene) have tendency to co-integrate into the same DSB site dramatically reducing frequency of usable events with site-specific trait gene insertions. Limiting delivered DNA molecules to donor DNA (for example, trait gene with homology arms) can increase the probability of events with desirable genotype. Therefore, the concept, described herein, of disrupted (inactive) endogenous or pre-integrated selectable marker genes that can be activated upon RGEN delivery, can make the DNA- and selectable marker-free approach for gene editing and gene insertion become very practical.

The guide polynucleotide can be introduced into a cell directly, as single stranded polynucleotide or a double stranded polynucleotide, using any method known in the art such as, but not limited to, particle bombardment, whiskers mediated transformation, Agrobacterium transformation or topical applications. The guide RNA can also be introduced indirectly into a cell by introducing a recombinant DNA molecule (via methods such as, but not limited to, particle bombardment or Agrobacterium transformation) comprising a heterologous nucleic acid fragment encoding a guide RNA, operably linked to a specific promoter that is capable of transcribing the guide RNA in said cell. The specific promoter can be, but is not limited to, a RNA polymerase Ill promoter, which allow for transcription of RNA with precisely defined, unmodified, 5′- and 3′-ends (DiCarlo et al., Nucleic Acids Res. 41: 4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3:e161). As described herein, direct delivery of a sgRNA into plant cells can be achieved through particle mediated delivery. Based on the experiments described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering gRNA in plant cells.

The guide polynucleotide can be produced by any method known in the art, including chemically synthesizing guide polynucleotides (such as but not limiting to Hendel et al. 2015, Nature Biotechnology 33, 985-989), in vitro generated guide polynucleotides, and/or self-splicing guide RNAs (such as but not limiting to Xie et al. 2015, PNAS 112:3570-3575).

The terms “target site”, “target sequence”, “target site sequence, “target DNA”, “target locus”, “genomic target site”, “genomic target sequence”, “genomic target locus” and “protospacer”, are used interchangeably herein and refer to a polynucleotide sequence such as, but not limited to, a nucleotide sequence on a chromosome, episome, a transgenic locus, or any other DNA molecule in the genome (including chromosomal, choloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which a guide polynucleotide/Cas endonuclease complex can recognize, bind to, and optionally nick or cleave. The target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature. As used herein, terms “endogenous target sequence” and “native target sequence” are used interchangeable herein to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cells as well as plants and seeds produced by the methods described herein. An “artificial target site” or “artificial target sequence” are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence can be identical in sequence to an endogenous or native target sequence in the genome of a cell but be located in a different position (i.e., a non-endogenous or non-native position) in the genome of a cell.

An “altered target site”, “altered target sequence”, “modified target site”, “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence. Such “alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

The length of the target DNA sequence (target site) can vary, and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. It is further possible that the target site can be palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary strand. The nick/cleavage site can be within the target sequence or the nick/cleavage site could be outside of the target sequence. In another variation, the cleavage could occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other Cases, the incisions could be staggered to produce single-stranded overhangs, also called “sticky ends”, which can be either 5′ overhangs, or 3′ overhangs. Active variants of genomic target sites can also be used. Such active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the given target site, wherein the active variants retain biological activity and hence are capable of being recognized and cleaved by an Cas endonuclease. Assays to measure the single or double-strand break of a target site by an endonuclease are known in the art and generally measure the overall activity and specificity of the agent on DNA substrates containing recognition sites.

A “protospacer adjacent motif” (PAM) herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide/Cas endonuclease system. The Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not followed by a PAM sequence. The sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used. The PAM sequence can be of any length but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long.

The terms “targeting”, “gene targeting” and “DNA targeting” are used interchangeably herein. DNA targeting herein may be the specific introduction of a knock-out, edit, or knock-in at a particular DNA sequence, such as in a chromosome or plasmid of a cell. In general, DNA targeting can be performed herein by cleaving one or both strands at a specific DNA sequence in a cell with a Cas protein associated with a suitable polynucleotide component. Such DNA cleavage, if a double-strand break (DSB), can prompt NHEJ or HDR processes which can lead to modifications at the target site.

The terms “knock-out”, “gene knock-out” and “genetic knock-out” are used interchangeably herein. A knock-out represents a DNA sequence of a cell that has been rendered partially or completely inoperative by targeting with a Cas protein; such a DNA sequence prior to knock-out could have encoded an amino acid sequence, or could have had a regulatory function (e.g., promoter), for example. A knock-out may be produced by an indel (insertion or deletion of nucleotide bases in a target DNA sequence through NHEJ), or by specific removal of sequence that reduces or completely destroys the function of sequence at or near the targeting site.

The guide polynucleotide/Cas endonuclease system can be used in combination with a co-delivered polynucleotide modification template to allow for editing (modification) of a genomic nucleotide sequence of interest. (See also U.S. Patent Application US 2015-0082478 A1, published on Mar. 19, 2015 and WO2015/026886 A1, published on Feb. 26, 2015, both are hereby incorporated in its entirety by reference.)

A “modified nucleotide” or “edited nucleotide” refers to a nucleotide sequence of interest that comprises at least one alteration when compared to its non-modified nucleotide sequence. Such “alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

The term “polynucleotide modification template” includes a polynucleotide that comprises at least one nucleotide modification when compared to the nucleotide sequence to be edited. A nucleotide modification can be at least one nucleotide substitution, addition or deletion. Optionally, the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

A polynucleotide modification template can be introduced into a cell by any method known in the art, such as, but not limited to, transient introduction methods, transfection, electroporation, microinjection, particle mediated delivery, topical application, whiskers mediated delivery, delivery via cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct delivery.

The polynucleotide modification template can be introduced into a cell as a single stranded polynucleotide molecule, a double stranded polynucleotide molecule, or as part of a circular DNA (vector DNA). The polynucleotide modification template can also be tethered to the guide RNA and/or the Cas endonuclease. Tethered DNAs can allow for co-localizing target and template DNA, useful in genome editing and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al. 2013 Nature Methods Vol. 10: 957-963.) The polynucleotide modification template may be present transiently in the cell or it can be introduced via a viral replicon.

The nucleotide to be edited can be located within or outside a target site recognized and cleaved by a Cas endonuclease. In one embodiment, the at least one nucleotide modification is not a modification at a target site recognized and cleaved by a Cas endonuclease. In another embodiment, there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900 or 1000 nucleotides between the at least one nucleotide to be edited and the genomic target site.

Genome editing can be accomplished using any method of gene editing available. For example, gene editing can be accomplished through the introduction into a host cell of a polynucleotide modification template (sometimes also referred to as a gene repair oligonucleotide) containing a targeted modification to a gene within the genome of the host cell. The polynucleotide modification template for use in such methods can be either single-stranded or double-stranded. Examples of such methods are generally described, for example, in US Publication No. 2013/0019349.

In one embodiment of the disclosure, the method comprises a method for modifying a nucleotide sequence in the genome of a cell, the method comprising: introducing into at least one cell comprising a target site and a disrupted selectable marker gene, at least one polynucleotide modification template, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein. The introducing and selection step does not comprise the introduction of a selectable marker gene, such as a recombinant DNA construct comprising a selectable marker gene. The disrupted selectable marker gene can be any disrupted marker gene including a disrupted visible marker gene.

Based on the experiments described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering a polynucleotide modification template in plant cells.

In some embodiments, gene editing may be facilitated through the induction of a double-stranded break (DSB) in a defined position in the genome near the desired alteration. DSBs can be induced using any DSB-inducing agent available, including, but not limited to, TALENs, meganucleases, zinc finger nucleases, nucleic acid guided-endonuclease systems, e.g. Cas9-gRNA systems (based on bacterial CRISPR-Cas systems), and the like. In some embodiments, the introduction of a DSB can be combined with the introduction of a polynucleotide modification template.

The process for editing a genomic sequence combining DSB and modification templates generally comprises: introducing to a host cell, a DSB-inducing agent, or a nucleic acid encoding a DSB-inducing agent, that recognizes a target sequence in the chromosomal sequence and is able to induce a DSB in the genomic sequence, and at least one polynucleotide modification template comprising at least one nucleotide alteration when compared to the nucleotide sequence to be edited. The polynucleotide modification template can further comprise nucleotide sequences flanking the at least one nucleotide alteration, in which the flanking sequences are substantially homologous to the chromosomal region flanking the DSB. Genome editing using DSB-inducing agents, such as Cas9-gRNA complexes, has been described, for example in U.S. Patent Application US 2015-0082478 A1, published on Mar. 19, 2015, WO2015/026886 A1, published on Feb. 26, 2015, U.S. application 62/023,246, filed on Jul. 7, 2014, and U.S. application 62/036,652, filed on Aug. 13, 2014, all of which are incorporated by reference herein.

Described herein are methods to method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template.

Gene editing using guided Cas endonucleases systems and a polynucleotide modification templates to modify a target sequence or a nucleotide of interest near a target sequence relies on homologous recombination/homologous directed repair (HDR) mechanisms that occur at a lower frequency in plant cell when compared to the frequency of Non Homologous End Joining (NHEJ). It would be desirable to increase the frequency of gene editing by not having to rely on HDR type mechanisms. Described herein are methods that enable gene editing without the use of a polynucleotide modification template, by relying on the restoration of a disrupted gene, wherein the restoration is accomplished by Non-Homologous-End-Joining (NHEJ) resulting in the insertion of at least a single base at the double strand break site or wherein the restoration is accomplished by the insertion of at least a single base at the double strand break site without the use of Homologous Recombination or Homology Directed Repair.

In one embodiment of the disclosure, the method comprises a method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising: a) introducing into at least one cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence; b) selecting a cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; and, c) introducing into a cell of (b) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and insert a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template.

In one embodiment of the disclosure, the method comprises a method for for editing a nucleotide sequence in the genome of a plant without the use of a polynucleotide modification template or donor DNA, the method comprising: a) introducing into at least one plant cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence; b) selecting a plant cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; c) regenerating a plant from the plant cell of (b); d) introducing into a cell from the plant of (c) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and inserting a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template; and, e) optimally, selecting a cell comprising the nucleotide insertion of (d).

The terms “knock-in”, “gene knock-in, “gene insertion” and “genetic knock-in” are used interchangeably herein. A knock-in represents the replacement or insertion of a DNA sequence at a specific DNA sequence in a cell by targeting with a Cas protein (by HR, wherein a suitable donor DNA polynucleotide is also used). Examples of knock-ins are a specific insertion of a heterologous amino acid coding sequence in a coding region of a gene, or a specific insertion of a transcriptional regulatory element in a genetic locus.

Various methods and compositions can be employed to obtain a cell or organism having a polynucleotide of interest inserted in a target site for a Cas endonuclease. Such methods can employ homologous recombination to provide integration of the polynucleotide of Interest at the target site. In one method provided, a polynucleotide of interest is provided to the organism cell in a donor DNA construct.

As used herein, “donor DNA” includes reference to a DNA construct that comprises a polynucleotide of interest to be inserted into the target site of a Cas endonuclease. The donor DNA construct can further comprise a first and a second region of homology that flank the polynucleotide of Interest. The first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome. The donor DNA can be tethered to the guide polynucleotide and/or the Cas endonuclease. Tethered donor DNAs can allow for co-localizing target and donor DNA, useful in genome editing and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al. 2013 Nature Methods Vol. 10:957-963.)

By “homology” is meant DNA sequences that are similar. For example, a “region of homology to a genomic region” that is found on the donor DNA is a region of DNA that has a similar sequence to a given “genomic region” in the cell or organism genome. A region of homology can be of any length that is sufficient to promote homologous recombination at the cleaved target site. For example, the region of homology can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100 or more bases in length such that the region of homology has sufficient homology to undergo homologous recombination with the corresponding genomic region. “Sufficient homology” indicates that two polynucleotide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction. The structural similarity includes overall length of each polynucleotide fragment, as well as the sequence similarity of the polynucleotides. Sequence similarity can be described by the percent sequence identity over the whole length of the sequences, and/or by conserved regions comprising localized similarities such as contiguous nucleotides having 100% sequence identity, and percent sequence identity over a portion of the length of the sequences.

The amount of homology or sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and/or regions having unit integral values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site. These ranges include every integer within the range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps. The amount of homology can also described by percent sequence identity over the full aligned length of the two polynucleotides which includes percent sequence identity of about at least 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus. Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, N.Y.); Current Protocols in Molecular Biology, Ausubel et al., Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, (Elsevier, New York).

As used herein, a “genomic region” is a segment of a chromosome in the genome of a cell that is present on either side of the target site or, alternatively, also comprises a portion of the target site. The genomic region can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800. 5-2900, 5-3000, 5-3100 or more bases such that the genomic region has sufficient homology to undergo homologous recombination with the corresponding region of homology.

Polynucleotides of interest and/or traits can be stacked together in a complex trait locus as described in US-2013-0263324-A1, published 3 Oct. 2013 and in PCT/US13/22891, published Jan. 24, 2013, both applications are hereby incorporated by reference. The guide polynucleotide/Cas9 endonuclease system described herein provides for an efficient system to generate double strand breaks and allows for traits to be stacked in a complex trait locus.

The guide polynucleotide/Cas endonuclease system can be used for introducing one or more polynucleotides of interest or one or more traits of interest into one or more target sites by introducing one or more guide polynucleotides, one Cas endonuclease, and optionally one or more donor DNAs to a plant cell. ((as described in US patent application US-2015-0082478-A1, published on Mar. 19, 2015, incorporated by reference herein). A fertile plant can be produced from that plant cell that comprises an alteration at said one or more target sites, wherein the alteration is selected from the group consisting of (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). Plants comprising these altered target sites can be crossed with plants comprising at least one gene or trait of interest in the same complex trait locus, thereby further stacking traits in said complex trait locus (see also US-2013-0263324-A1, published Oct. 3, 2013 and in PCT/US13/22891, published Jan. 24, 2013, incorporated by reference herein).

The structural similarity between a given genomic region and the corresponding region of homology found on the donor DNA can be any degree of sequence identity that allows for homologous recombination to occur. For example, the amount of homology or sequence identity shared by the “region of homology” of the donor DNA and the “genomic region” of the organism genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination

The region of homology on the donor DNA can have homology to any sequence flanking the target site. While in some embodiments the regions of homology share significant sequence homology to the genomic sequence immediately flanking the target site, it is recognized that the regions of homology can be designed to have sufficient homology to regions that may be further 5′ or 3′ to the target site. In still other embodiments, the regions of homology can also have homology with a fragment of the target site along with downstream genomic regions. In one embodiment, the first region of homology further comprises a first fragment of the target site and the second region of homology comprises a second fragment of the target site, wherein the first and second fragments are dissimilar.

Once a double-strand break is induced in the DNA, the cell's DNA repair mechanism is activated to repair the break. The Non-Homologous-End-Joining (NHEJ) pathways are the most common repair mechanism to bring the broken ends together (Bleuyard et al., (2006) DNA Repair 5:1-12). The structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible. The two ends of one double-strand break are the most prevalent substrates of NHEJ (Kirik et al., (2000) EMBO J 19:5562-6), however if two different double-strand breaks occur, the free ends from different breaks can be ligated and result in chromosomal deletions (Siebert and Puchta, (2002) Plant Cell 14:1121-31), or chromosomal translocations between different chromosomes (Pacher et al., (2007) Genetics 175:21-9). Error-prone DNA repair mechanisms can produce mutations at double-strand break sites. The Non-Homologous-End-Joining (NHEJ) pathways are the most common repair mechanism to bring the broken ends together (Bleuyard et al., (2006) DNA Repair 5:1-12).

Alternatively, the double-strand break can be repaired by homologous recombination (HR) between homologous DNA sequences. Once the sequence around the double-strand break is altered, for example, by exonuclease activities involved in the maturation of double-strand breaks, gene conversion pathways can restore the original structure if a homologous sequence is available, such as a homologous chromosome in non-dividing somatic cells, or a sister chromatid after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and/or epigenic DNA sequences may also serve as a DNA repair template for homologous recombination (Puchta, (1999) Genetics 152:1173-81). Episomal DNA molecules can also be ligated into the double-strand break, for example, integration of T-DNAs into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta, (1998) EMBO J 17:6086-95).

As used herein, “homologous recombination (HR)” includes the exchange of DNA fragments between two DNA molecules at the sites of homology. The frequency of homologous recombination is influenced by a number of factors. Different organisms vary with respect to the amount of homologous recombination and the relative proportion of homologous to non-homologous recombination. Generally, the length of the region of homology affects the frequency of homologous recombination events: the longer the region of homology, the greater the frequency. The length of the homology region needed to observe homologous recombination is also species-variable. In many cases, at least 5 kb of homology has been utilized, but homologous recombination has been observed with as little as 25-50 bp of homology. See, for example, Singer et al., (1982) Cell 31:25-33; Shen and Huang, (1986) Genetics 112:441-57; Watt et al., (1985) Proc. Natl. Acad. Sci. USA 82:4768-72, Sugawara and Haber, (1992) Mol Cell Biol 12:563-75, Rubnitz and Subramani, (1984) Mol Cell Biol 4:2253-8; Ayares et al., (1986) Proc. Natl. Acad. Sci. USA 83:5199-203; Liskay et al., (1987) Genetics 115:161-7.

Homology-directed repair (HDR) is a mechanism in cells to repair double-stranded and single stranded DNA breaks. Homology-directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. 79:181-211). The most common form of HDR is called homologous recombination (HR), which has the longest sequence homology requirements between the donor and acceptor DNA. Other forms of HDR include single-stranded annealing (SSA) and breakage-induced replication, and these require shorter sequence homology relative to HR. Homology-directed repair at nicks (single-stranded breaks) can occur via a mechanism distinct from HDR at double-strand breaks (Davis and Maizels. PNAS (0027-8424), 111 (10), p. E924-E932.

Alteration of the genome of a plant cell, for example, through homology-directed repair (HDR), is a powerful tool for genetic engineering. Despite the low frequency of homologous recombination in higher plants, there are a few examples of successful homologous recombination of plant endogenous genes. The parameters for homologous recombination in plants have primarily been investigated by rescuing introduced truncated selectable marker genes. In these experiments, the homologous DNA fragments were typically between 0.3 kb to 2 kb. Observed frequencies for homologous recombination were on the order of 10⁻⁴ to 10⁻⁵. See, for example, Halfter et al., (1992) Mol Gen Genet 231:186-93; Offringa et al., (1990) EMBO J 9:3077-84; Offringa et al., (1993) Proc. Natl. Acad. Sci. USA 90:7346-50; Paszkowski et al., (1988) EMBO J 7:4021-6; Hourda and Paszkowski, (1994) Mol Gen Genet 243:106-11; and Risseeuw et al., (1995) Plant J 7:109-19.

DNA double-strand breaks appear to be an effective factor to stimulate homologous recombination pathways (Puchta et al., (1995) Plant Mol Biol 28:281-92; Tzfira and White, (2005) Trends Biotechnol 23:567-9; Puchta, (2005) J Exp Bot 56:1-14). Using DNA-breaking agents, a two- to nine-fold increase of homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al., (1995) Plant Mol Biol 28:281-92). In maize protoplasts, experiments with linear DNA molecules demonstrated enhanced homologous recombination between plasmids (Lyznik et al., (1991) Mol Gen Genet 230:209-18).

The donor DNA may be introduced by any means known in the art. For example, a plant having a target site is provided. The donor DNA may be provided by any delivery method known in the art including, for example, Agrobacterium-mediated transformation, whiskers mediated transformation, or biolistic particle bombardment. The donor DNA may be present transiently in the cell or it can be introduced via a viral replicon. In the presence of the Cas endonuclease and the target site, the donor DNA is inserted into the plant's genome.

As described herein, direct delivery of a donor DNA into plant cells can be achieved through particle mediated delivery. Based on the experiments described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, electroporation, particle bombardment, whiskers mediated delivery, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering a donor DNA in plant cells.

In one embodiment of the disclosure, the method comprises a method for modifying a nucleotide sequence in the genome of a cell, the method comprising: introducing into at least one cell comprising a target site and a disrupted selectable marker gene, at least one donor DNA, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein. The introducing and selection step does not comprise the introduction of a selectable marker gene, such as a recombinant DNA construct comprising a selectable marker gene. The disrupted selectable marker gene can be any disrupted marker gene including a disrupted visible marker gene.

Further uses for guide RNA/Cas endonuclease systems have been described (See U.S. Patent Application US 2015-0082478 A1, published on Mar. 19, 2015, WO2015/026886 A1, published on Feb. 26, 2015, US 2015-0059010 A1, published on Feb. 26, 2015, U.S. application 62/023,246, filed on Jul. 7, 2014, and U.S. application 62/036,652, filed on Aug. 13, 2014, all of which are incorporated by reference herein) and include but are not limited to modifying or replacing nucleotide sequences of interest (such as a regulatory elements), insertion of polynucleotides of interest, gene knock-out, gene-knock in, modification of splicing sites and/or introducing alternate splicing sites, modifications of nucleotide sequences encoding a protein of interest, amino acid and/or protein fusions, and gene silencing by expressing an inverted repeat into a gene of interest.

Polynucleotides of interest are further described herein and include polynucleotides reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for genetic engineering will change accordingly.

Further provided are methods for identifying at least one plant cell, comprising in its genome, a polynucleotide of interest integrated at the target site. A variety of methods are available for identifying those plant cells with insertion into the genome at or near to the target site without using a screenable marker phenotype. Such methods can be viewed as directly analyzing a target sequence to detect any change in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blots, and any combination thereof. See, for example, U.S. patent application Ser. No. 12/147,834, herein incorporated by reference to the extent necessary for the methods described herein. The method also comprises recovering a plant from the plant cell comprising a polynucleotide of Interest integrated into its genome. The plant may be sterile or fertile. It is recognized that any polynucleotide of interest can be provided, integrated into the plant genome at the target site, and expressed in a plant.

Polynucleotides/polypeptides of interest include, but are not limited to, herbicide-resistance coding sequences, insecticidal coding sequences, nematicidal coding sequences, antimicrobial coding sequences, antifungal coding sequences, antiviral coding sequences, abiotic and biotic stress tolerance coding sequences, or sequences modifying plant traits such as yield, grain quality, nutrient content, starch quality and quantity, nitrogen fixation and/or utilization, fatty acids, and oil content and/or composition. More specific polynucleotides of interest include, but are not limited to, genes that improve crop yield, polypeptides that improve desirability of crops, genes encoding proteins conferring resistance to abiotic stress, such as drought, nitrogen, temperature, salinity, toxic metals or trace elements, or those conferring resistance to toxins such as pesticides and herbicides, or to biotic stress, such as attacks by fungi, viruses, bacteria, insects, and nematodes, and development of diseases associated with these organisms. General categories of genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, fertility or sterility, grain characteristics, and commercial products. Genes of interest include, generally, those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting kernel size, sucrose loading, and the like that can be stacked or used in combination with other traits, such as but not limited to herbicide resistance, described herein.

Agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, introducing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated by reference.

Polynucleotide sequences of interest may encode proteins involved in introducing disease or pest resistance. By “disease resistance” or “pest resistance” is intended that the plants avoid the harmful symptoms that are the outcome of the plant-pathogen interactions. Pest resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Disease resistance and insect resistance genes such as lysozymes or cecropins for antibacterial protection, or proteins such as defensins, glucanases or chitinases for antifungal protection, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, or glycosidases for controlling nematodes or insects are all examples of useful gene products. Genes encoding disease resistance traits include detoxification genes, such as against fumonisin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); and the like. Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); and the like.

An “herbicide resistance protein” or a protein resulting from expression of an “herbicide resistance-encoding nucleic acid molecule” includes proteins that confer upon a cell the ability to tolerate a higher concentration of an herbicide than cells that do not express the protein, or to tolerate a certain concentration of an herbicide for a longer period of time than cells that do not express the protein. Herbicide resistance traits may be introduced into plants by genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS also called AHAS), in particular the sulfonylurea-type (UK: sulphonylurea) herbicides, genes coding for resistance to herbicides that act to inhibit the action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), glyphosate (e.g., the EPSP synthase gene and the GAT gene), HPPD inhibitors (e.g, the HPPD gene) or other such genes known in the art. See, for example, U.S. Pat. Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and U.S. Provisional Application No. 61/401,456, each of which is herein incorporated by reference. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutants encode resistance to the herbicide chlorsulfuron.

As used herein, a “sulfonylurea-tolerant polypeptide” comprises any polypeptide which when expressed in a plant confers tolerance to at least one sulfonylurea. Sulfonylurea herbicides inhibit growth of higher plants by blocking acetolactate synthase (ALS), also known as, acetohydroxy acid synthase (AHAS). Plants containing particular mutations in ALS (e.g., the S4 and/or HRA mutations) are tolerant to sulfonylurea herbicides. The production of sulfonylurea-tolerant plants is described more fully in U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; and international publication WO 96/33270, which are incorporated herein by reference in their entireties for all purposes, and in Tan et al. 2005. Imidazolinone-tolerant crops: history, current status and future. Pest Manag Sci 61:246-257. The sulfonylurea-tolerant polypeptide can be encoded by, for example, the SuRA or SuRB locus of ALS. In specific embodiments, the ALS inhibitor-tolerant polypeptide comprises the C3 ALS mutant, the HRA ALS mutant, the S4 mutant or the S4/HRA mutant or any combination thereof. Different mutations in ALS are known to confer tolerance to different herbicides and groups (and/or subgroups) of herbicides; see, e.g., Tranel and Wright (2002) Weed Science 50:700-712. See also, U.S. Pat. Nos. 5,605,011, 5,378,824, 5,141,870, and 5,013,659, each of which is herein incorporated by reference in their entirety. The HRA mutation in ALS finds particular use in one embodiment. The mutation results in the production of an acetolactate synthase polypeptide which is resistant to at least one sulfonylurea compound in

A gene encoding a sulfonylurea-tolerant polypeptide is referred to as a sulfonyl tolerant gene or a sulfonyl resistant gene. The terms sulfonyl tolerant gene or sulfonyl resistant gene are used interchangeably herein.

A disrupted sulfonylurea resistant (ALS) gene refers to a disrupted gene, of which its corresponding undisrupted gene encodes a sulfonylurea-tolerant polypeptide, that is modified such that its gene product no longer encodes a functional sulfonylurea-tolerant polypeptide.

In one embodiment, the method comprises a method for producing a sulfonylurea resistant plant comprising a modified target site, the method comprising: a) introducing into a plant cell comprising a disrupted sulfonylurea resistant (ALS) gene, a first guide RNA, a Cas9 endonuclease, at least a second guide RNA, wherein said first guide RNA and Cas9 endonuclease can form a first complex capable of introducing a double strand in said disrupted sulfonylurea resistant (ALS) gene, wherein said second guide RNA and Cas9 endonuclease can form a second complex capable of introducing a double strand break at said target site; and, b) obtaining a sulfonylurea resistant plant from said plant cell, wherein said sulfonylurea resistant plant comprises a modification at said target, wherein said modification is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).

Components of a sulfonylurea-responsive repressor system (as described in U.S. Pat. No. 8,257,956, issued on Sep. 4, 2012) can also be introduced into plant genomes to generate a repressor/operator/inducer systems into said plant where polypeptides can specifically bind to an operator, wherein the specific binding is regulated by a sulfonylurea compound.

Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male fertility genes such as MS26 (see for example U.S. Pat. Nos. 7,098,388, 7,517,975, 7,612,251), MS45 (see for example U.S. Pat. Nos. 5,478,369, 6,265,640) or MSCA1 (see for example U.S. Pat. No. 7,919,676). Maize plants (Zea mays L.) can be bred by both self-pollination and cross-pollination techniques. Maize has male flowers, located on the tassel, and female flowers, located on the ear, on the same plant. It can self-pollinate (“selfing”) or cross pollinate. Natural pollination occurs in maize when wind blows pollen from the tassels to the silks that protrude from the tops of the incipient ears. Pollination may be readily controlled by techniques known to those of skill in the art. The development of maize hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selections are two of the breeding methods used to develop inbred lines from populations. Breeding programs combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. A hybrid maize variety is the cross of two such inbred lines, each of which may have one or more desirable characteristics lacked by the other or which complement the other. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which have commercial potential. The hybrid progeny of the first generation is designated F1. The F1 hybrid is more vigorous than its inbred parents. This hybrid vigor, or heterosis, can be manifested in many ways, including increased vegetative growth and increased yield.

Hybrid maize seed can be produced by a male sterility system incorporating manual detasseling. To produce hybrid seed, the male tassel is removed from the growing female inbred parent, which can be planted in various alternating row patterns with the male inbred parent. Consequently, introducing that there is sufficient isolation from sources of foreign maize pollen, the ears of the female inbred will be fertilized only with pollen from the male inbred. The resulting seed is therefore hybrid (F1) and will form hybrid plants.

Field variation impacting plant development can result in plants tasseling after manual detasseling of the female parent is completed. Or, a female inbred plant tassel may not be completely removed during the detasseling process. In any event, the result is that the female plant will successfully shed pollen and some female plants will be self-pollinated. This will result in seed of the female inbred being harvested along with the hybrid seed which is normally produced. Female inbred seed does not exhibit heterosis and therefore is not as productive as F1 seed. In addition, the presence of female inbred seed can represent a germplasm security risk for the company producing the hybrid.

Alternatively, the female inbred can be mechanically detasseled by machine. Mechanical detasseling is approximately as reliable as hand detasseling, but is faster and less costly. However, most detasseling machines produce more damage to the plants than hand detasseling. Thus, no form of detasseling is presently entirely satisfactory, and a need continues to exist for alternatives which further reduce production costs and to eliminate self-pollination of the female parent in the production of hybrid seed.

Mutations that cause male sterility in plants have the potential to be useful in methods for hybrid seed production for crop plants such as maize and can lower production costs by eliminating the need for the labor-intensive removal of male flowers (also known as de-tasseling) from the maternal parent plants used as a hybrid parent. Mutations that cause male sterility in maize have been produced by a variety of methods such as X-rays or UV-irradiations, chemical treatments, or transposable element insertions (ms23, ms25, ms26, ms32) (Chaubal et al. (2000) Am J Bot 87:1193-1201). Conditional regulation of fertility genes through fertility/sterility “molecular switches” could enhance the options for designing new male-sterility systems for crop improvement (Unger et al. (2002) Transgenic Res 11:455-465).

Furthermore, it is recognized that the polynucleotide of interest may also comprise antisense sequences complementary to at least a portion of the messenger RNA (mRNA) for a targeted gene sequence of interest. Antisense nucleotides are constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA. In this manner, antisense constructions having 70%, 80%, or 85% sequence identity to the corresponding antisense sequences may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used.

In addition, the polynucleotide of interest may also be used in the sense orientation to suppress the expression of endogenous genes in plants. Methods for suppressing gene expression in plants using polynucleotides in the sense orientation are known in the art. The methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, generally greater than about 65% sequence identity, about 85% sequence identity, or greater than about 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323; herein incorporated by reference.

The polynucleotide of interest can also be a phenotypic marker.

The recombinant DNA molecules, DNA sequences of interest, and polynucleotides of interest can comprise one or more DNA sequences for gene silencing. Methods for gene silencing involving the expression of DNA sequences in plant are known in the art include, but are not limited to, cosuppression, antisense suppression, double-stranded RNA (dsRNA) interference, hairpin RNA (hpRNA) interference, intron-containing hairpin RNA (ihpRNA) interference, transcriptional gene silencing, and micro RNA (miRNA) interference

As used herein, “nucleic acid” means a polynucleotide and includes a single or a double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Thus, the terms “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence” and “nucleic acid fragment” are used interchangeably to denote a polymer of RNA and/or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenosine or deoxyadenosine (for RNA or DNA, respectively), “C” for cytosine or deoxycytosine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

“Open reading frame” is abbreviated ORF.

The terms “subfragment that is functionally equivalent” and “functionally equivalent subfragment” are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used in the design of genes to produce the desired phenotype in a transformed plant. Genes can be designed for use in suppression by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the sense or antisense orientation relative to a plant promoter sequence.

The term “conserved domain” or “motif” means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential to the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or “signatures”, to determine if a protein with a newly determined sequence belongs to a previously identified protein family.

Polynucleotide and polypeptide sequences, variants thereof, and the structural relationships of these sequences can be described by the terms “homology”, “homologous”, “substantially identical”, “substantially similar” and “corresponding substantially” which are used interchangeably herein. These refer to polypeptide or nucleic acid fragments wherein changes in one or more amino acids or nucleotide bases do not affect the function of the molecule, such as the ability to mediate gene expression or to produce a certain phenotype. These terms also refer to modification(s) of nucleic acid fragments that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. These modifications include deletion, substitution, and/or insertion of one or more nucleotides in the nucleic acid fragment.

Substantially similar nucleic acid sequences encompassed may be defined by their ability to hybridize (under moderately stringent conditions, e.g., 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.

The term “selectively hybridizes” includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.

The term “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a probe will selectively hybridize to its target sequence in an in vitro hybridization assay. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salt(s)) at pH 7.0 to 8.3, and at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2× SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

“Sequence identity” or “identity” in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

The term “percentage of sequence identity” refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any integer percentage from 50% to 100%. These identities can be determined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

The “Clustal W method of alignment” corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs (%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using a GAP creation penalty weight of 8 and a gap length extension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915). GAP uses the algorithm of Needleman and Wunsch, (1970) J Mol Biol 48:443-53, to find an alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps, using a gap creation penalty and a gap extension penalty in units of matched bases.

“BLAST” is a searching algorithm provided by the National Center for Biotechnology Information (NCBI) used to find regions of similarity between biological sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches to identify sequences having sufficient similarity to a query sequence such that the similarity would not be predicted to have occurred randomly. BLAST reports the identified sequences and their local alignment to the query sequence.

It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides from other species or modified naturally or synthetically wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any integer percentage from 50% to 100%. Indeed, any integer amino acid identity from 50% to 100% may be useful in describing the present disclosure, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

“Gene” includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences.

A “mutated gene” is a gene that has been altered through human intervention. Such a “mutated gene” has a sequence that differs from the sequence of the corresponding non-mutated gene by at least one nucleotide addition, deletion, or substitution. In certain embodiments of the disclosure, the mutated gene comprises an alteration that results from a guide polynucleotide/Cas endonuclease system as disclosed herein. A mutated plant is a plant comprising a mutated gene.

As used herein, a “targeted mutation” is a mutation in a native gene that was made by altering a target sequence within the native gene using a method involving a double-strand-break-inducing agent that is capable of inducing a double-strand break in the DNA of the target sequence as disclosed herein or known in the art.

The guide RNA/Cas endonuclease induced targeted mutation can occur in a nucleotide sequence that is located within or outside a genomic target site that is recognized and cleaved by a Cas endonuclease.

The term “genome” as it applies to a plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondria, or plastid) of the cell.

A “codon-modified gene” or “codon-preferred gene” or “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.

An “allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same, that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ, that plant is heterozygous at that locus.

“Coding sequence” refers to a polynucleotide sequence which codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to: promoters, translation leader sequences, 5′ untranslated sequences, 3′ untranslated sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures.

“A plant-optimized nucleotide sequence” is nucleotide sequence that has been optimized for increased expression in plants. For example, a plant-optimized nucleotide sequence can be synthesized by modifying a nucleotide sequence encoding a protein such as, for example, double-strand-break-inducing agent (e.g., an endonuclease) as disclosed herein, using one or more plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage.

Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Additional sequence modifications are known to enhance gene expression in a plant host. These include, for example, elimination of: one or more sequences encoding spurious polyadenylation signals, one or more exon-intron splice site signals, one or more transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given plant host, as calculated by reference to known genes expressed in the host plant cell. When possible, the sequence is modified to avoid one or more predicted hairpin secondary mRNA structures. Thus, “a plant-optimized nucleotide sequence” of the present disclosure comprises one or more of such sequence modifications.

A promoter is a region of DNA involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. An “enhancer” is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, and/or comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.

It has been shown that certain promoters are able to direct RNA synthesis at a higher rate than others. These are called “strong promoters”. Certain other promoters have been shown to direct RNA synthesis at higher levels only in particular types of cells or tissues and are often referred to as “tissue specific promoters”, or “tissue-preferred promoters” if the promoters direct RNA synthesis preferably in certain tissues but also in other tissues at reduced levels. Since patterns of expression of a chimeric gene (or genes) introduced into a plant are controlled using promoters, there is an ongoing interest in the isolation of novel promoters which are capable of controlling the expression of a chimeric gene or (genes) at certain levels in specific tissue types or at specific plant developmental stages.

A plant promoter can include a promoter capable of initiating transcription in a plant cell, for a review of plant promoters, see, Potenza et al., (2004) In Vitro Cell Dev Biol 40:1-22. Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al., (1985) Nature 313:810-2); rice actin (McElroy et al., (1990) Plant Cell 2:163-71); ubiquitin (Christensen et al., (1989) Plant Mol Biol 12:619-32; Christensen et al., (1992) Plant Mol Biol 18:675-89); pEMU (Last et al., (1991) Theor Appl Genet 81:581-8); MAS (Velten et al., (1984) EMBO J 3:2723-30); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters are described in, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611. In some examples an inducible promoter may be used. Pathogen-inducible promoters induced following infection by a pathogen include, but are not limited to those regulating expression of PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. The promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters include, but are not limited to, the maize In2-2 promoter, activated by benzene sulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-II-27, WO93/01294), activated by hydrophobic electrophilic compounds used as pre-emergent herbicides, and the tobacco PR-1a promoter (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid. Other chemical-regulated promoters include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter (Schena et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-5; McNellis et al., (1998) Plant J 14:247-257); tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156).

Tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue. Tissue-preferred promoters include, for example, Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Hansen et al., (1997) Mol Gen Genet 254:337-43; Russell et al., (1997) Transgenic Res 6:157-68; Rinehart et al., (1996) Plant Physiol 112:1331-41; Van Camp et al., (1996) Plant Physiol 112:525-35; Canevascini et al., (1996) Plant Physiol 112:513-524; Lam, (1994) Results Probl Cell Differ 20:181-96; and Guevara-Garcia et al., (1993) Plant J 4:495-505. Leaf-preferred promoters include, for example, Yamamoto et al., (1997) Plant J 12:255-65; Kwon et al., (1994) Plant Physiol 105:357-67; Yamamoto et al., (1994) Plant Cell Physiol 35:773-8; Gotor et al., (1993) Plant J 3:509-18; Orozco et al., (1993) Plant Mol Biol 23:1129-38; Matsuoka et al., (1993) Proc. Natl. Acad. Sci. USA 90:9586-90; Simpson et al., (1958) EMBO J 4:2723-9; Timko et al., (1988) Nature 318:57-8. Root-preferred promoters include, for example, Hire et al., (1992) Plant Mol Biol 20:207-18 (soybean root-specific glutamine synthase gene); Miao et al., (1991) Plant Cell 3:11-22 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol Biol 14:433-43 (root-specific promoter of A. tumefaciens mannopine synthase (MAS)); Bogusz et al., (1990) Plant Cell 2:633-41 (root-specific promoters isolated from Parasponia andersonii and Trema tomentosa); Leach and Aoyagi, (1991) Plant Sci 79:69-76 (A. rhizogenes roIC and roID root-inducing genes); Teeri et al., (1989) EMBO J 8:343-50 (Agrobacterium wound-induced TR1′ and TR2′ genes); VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol Biol 29:759-72); and roIB promoter (Capana et al., (1994) Plant Mol Biol 25:681-91; phaseolin gene (Murai et al., (1983) Science 23:476-82; Sengopta-Gopalen et al., (1988) Proc. Natl. Acad. Sci. USA 82:3320-4). See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179.

Seed-preferred promoters include both seed-specific promoters active during seed development, as well as seed-germinating promoters active during seed germination. See, Thompson et al., (1989) BioEssays 10:108. Seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1-phosphate synthase); (WO00/11177; and U.S. Pat. No. 6,225,529). For dicots, seed-preferred promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-preferred promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, and nuc1. See also, WO00/12733, where seed-preferred promoters from END1 and END2 genes are disclosed.

The term “inducible promoter” refers to promoters that selectively express a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters induced or regulated by light, heat, stress, flooding or drought, salt stress, osmotic stress, phytohormones, wounding, or chemicals such as ethanol, abscisic acid (ABA), jasmonate, salicylic acid, or safeners.

An example of a stress-inducible is RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91). One of ordinary skill in the art is familiar with protocols for simulating drought conditions and for evaluating drought tolerance of plants that have been subjected to simulated or naturally-occurring drought conditions. For example, one can simulate drought conditions by giving plants less water than normally required or no water over a period of time, and one can evaluate drought tolerance by looking for differences in physiological and/or physical condition, including (but not limited to) vigor, growth, size, or root length, or in particular, leaf color or leaf area size. Other techniques for evaluating drought tolerance include measuring chlorophyll fluorescence, photosynthetic rates and gas exchange rates. Also, one of ordinary skill in the art is familiar with protocols for simulating stress conditions such as osmotic stress, salt stress and temperature stress and for evaluating stress tolerance of plants that have been subjected to simulated or naturally-occurring stress conditions.

Another example of an inducible promoter useful in plant cells has been described in US patent application, US 2013-0312137A1, published on Nov. 21, 2013, incorporated by reference herein. US patent application US 2013-0312137A1 describes a ZmCAS1 promoter from a CBSU-Anther_Subtraction library (CAS1) gene encoding a mannitol dehydrogenase from maize, and functional fragments thereof. The ZmCAS1 promoter (also referred to as “CAS1 promoter”, “mannitol dehydrogenase promoter”, “mdh promoter”) can be induced by a chemical or stress treatment. The chemical can be a safener such as, but not limited to, N-(aminocarbonyl)-2-chlorobenzenesulfonamide (2-CBSU). The stress treatment can be a heat treatment such as, but not limited to, a heat shock treatment (see also U.S. provisional patent application, 62/120,421, filed on Feb. 25, 2015, and incorporated by reference herein.

New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, (1989) In The Biochemistry of Plants, Vol. 115, Stumpf and Conn, eds (New York, N.Y.: Academic Press), pp. 1-82.

“Translation leader sequence” refers to a polynucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (e.g., Turner and Foster, (1995) Mol Biotechnol 3:225-236).

“3′ non-coding sequences”, “transcription terminator” or “termination sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 1:671-680.

“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complimentary copy of the DNA sequence, it is referred to as the primary transcript or pre-mRNA. A RNA transcript is referred to as the mature RNA or mRNA when it is a RNA sequence derived from post-transcriptional processing of the primary transcript pre mRNA. “Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell. “crDNA” refers to a DNA that is complementary to, and synthesized from, an mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into double-stranded form using the Klenow fragment of DNA polymerase I. “Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (see, e.g., U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes. The terms “complement” and “reverse complement” are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions can be operably linked, either directly or indirectly, 5′ to the target mRNA, or 3′ to the target mRNA, or within the target mRNA, or a first complementary region is 5′ and its complement is 3′ to the target mRNA.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989). Transformation methods are well known to those skilled in the art and are described infra.

“PCR” or “polymerase chain reaction” is a technique for the synthesis of specific DNA segments and consists of a series of repetitive denaturation, annealing, and extension cycles. Typically, a double-stranded DNA is heat denatured, and two primers complementary to the 3′ boundaries of the target segment are annealed to the DNA at low temperature, and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a “cycle”.

The term “recombinant” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis, or manipulation of isolated segments of nucleic acids by genetic engineering techniques.

The terms “plasmid”, “vector” and “cassette” refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of double-stranded DNA. Such elements may be autonomously replicating sequences, genome integrating sequences, phage, or nucleotide sequences, in linear or circular form, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a polynucleotide of interest into a cell. “Transformation cassette” refers to a specific vector containing a gene and having elements in addition to the gene that facilitates transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a gene and having elements in addition to the gene that allow for expression of that gene in a host.

The terms “recombinant DNA molecule”, “recombinant construct”, “expression construct”, “construct”, “construct”, and “recombinant DNA construct” are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not all found together in nature. For example, a construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells. The skilled artisan will also recognize that different independent transformation events may result in different levels and patterns of expression (Jones et al., (1985) EMBO J 4:2411-2418; De Almeida et al., (1989) Mol Gen Genetics 218:78-86), and thus that multiple events are typically screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished standard molecular biological, biochemical, and other assays including Southern analysis of DNA, Northern analysis of mRNA expression, PCR, real time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting analysis of protein expression, enzyme or activity assays, and/or phenotypic analysis.

The term “expression”, as used herein, refers to the production of a functional end-product (e.g., an mRNA, guide RNA, or a protein) in either precursor or mature form.

The term “introducing” includes reference to introducing, providing, contacting a compound, such as but not limited to, a nucleic acid (e.g., expression construct) or peptide, polypeptide or protein into a cell. Introducing includes the direct delivery of polynucleotides (such as RNA, DNA, RNA-DNA hibrids, single or double stranded oligonucleotides, linear or circular polynucleotides) and/or includes the direct delivery of proteins (polypeptides). Introducing includes reference to the incorporation of a nucleic acid or polypeptide into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient introduction of a nucleic acid or protein into the cell. Introducing includes reference to stable or transient transformation methods, transfection, transduction, microinjection, electroporation, viral methods, Agrobacterium-mediated transformation, ballistic particle acceleration, whiskers mediated transformation, as well as sexually crossing. Thus, “introducing” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct, guide RNA, guide DNA, template DNA, donor DNA) into a cell, includes “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

A variety of methods are known for introducing, contacting and/or providing a composition into an organisms including stable transformation methods, transient transformation methods, virus-mediated methods, sexual crossing and sexual breeding. Stable transformation indicates that the introduced polynucleotide integrates into the genome of the organism and is capable of being inherited by progeny thereof. Transient transformation indicates that the introduced composition is only temporarily expressed or present in the organism.

Protocols for contacting, providing, introducing polynucleotides and polypeptides into cells or organisms are known and include microinjection (Crossway et al., (1986) Biotechniques 4:320-34 and U.S. Pat. No. 6,300,543), meristem transformation (U.S. Pat. No. 5,736,369), electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-6, Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), whiskers mediated transformation (Ainley et al. 2013, Plant Biotechnology Journal 11:1126-1134; Shaheen A. and M. Arshad 2011 Properties and Applications of Silicon Carbide (2011), 345-358 Editor(s): Gerhardt, Rosario. Publisher: InTech, Rijeka, Croatia. CODEN: 69PQBP; ISBN: 978-953-307-201-2) direct gene transfer (Paszkowski et al., (1984) EMBO J 3:2717-22), and ballistic particle acceleration (U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al., (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg & Phillips (Springer-Verlag, Berlin); McCabe et al., (1988) Biotechnology 6:923-6; Weissinger et al., (1988) Ann Rev Genet 22:421-77; Sanford et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al., (1988) Plant Physiol 87:671-4 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev Biol 27P:175-82 (soybean); Singh et al., (1998) Theor Appl Genet 96:319-24 (soybean); Datta et al., (1990) Biotechnology 8:736-40 (rice); Klein et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-9 (maize); Klein et al., (1988) Biotechnology 6:559-63 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol 91:440-4 (maize); Fromm et al., (1990) Biotechnology 8:833-9 (maize); Hooykaas-Van Slogteren et al., (1984) Nature 311:763-4; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-9 (Liliaceae); De Wet et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Rep 9:415-8) and Kaeppler et al., (1992) Theor Appl Genet 84:560-6 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4:1495-505 (electroporation); Li et al., (1993) Plant Cell Rep 12:250-5; Christou and Ford (1995) Annals Botany 75:407-13 (rice) and Osjoda et al., (1996) Nat Biotechnol 14:745-50 (maize via Agrobacterium tumefaciens).

Alternatively, polynucleotides may be introduced into cells or organisms by contacting cells or organisms with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide within a viral DNA or RNA molecule. In some examples a polypeptide of interest may be initially synthesized as part of a viral polyprotein, which is later processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known, see, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931. Transient transformation methods include, but are not limited to, the introduction of polypeptides, such as a double-strand break inducing agent, directly into the organism, the introduction of polynucleotides such as DNA and/or RNA polynucleotides, and the introduction of the RNA transcript, such as an mRNA encoding a double-strand break inducing agent, into the organism. Such methods include, for example, microinjection or particle bombardment. See, for example Crossway et al., (1986) Mol Gen Genet 202:179-85; Nomura et al., (1986) Plant Sci 44:53-8; Hepler et al., (1994) Proc. Natl. Acad. Sci. USA 91:2176-80; and, Hush et al., (1994) J Cell Sci 107:775-84.

Nucleic acids and proteins can be provided to a cell by any method including methods using molecules to facilitate the uptake of anyone or all components of a guided Cas system (protein and/or nucleic acids), such as cell-penetrating peptides and nanocarriers. See also US20110035836 Nanocarrier based plant transfection and transduction, and EP 2821486 A1 Method of introducing nucleic acid into plant cells, incorporated herein by reference.

Introducing a guide RNA/Cas endonuclease complex into a cell includes introducing the individual components of said complex either separately or combined into the cell, and either directly (direct delivery as RNA for the guide and protein for the Cas endonuclease) or via recombination constructs expressing the components (guide RNA, Cas endonuclease). Introducing a guide RNA/Cas endonuclease complex into a cell includes introducing the guide RNA/Cas endonuclease complex as a ribonucleotide-protein into the cell. The ribonucleotide-protein can be assembled prior to being introduced into the cell as described herein.

Plant cells differ from human and animal cells in that plant cells contain a plant cell wall which may act as a barrier to the direct delivery of the RGEN ribonucleoproteins and/or of the direct delivery of the RGEN components.

As described herein, direct delivery of the RGEN ribonucleoproteins into plant cells can be achieved through particle mediated delivery (particle bombardment. Based on the experiments described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, electroporation, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, can be successfully used for delivering RGEN ribonucleoproteins into plant cells.

Direct delivery of the RGEN ribonucleoprotein, as described herein, allows for genome editing at a target site in the genome of a cell which can be followed by rapid degradation of the complex, and only a transient presence of the complex in the cell. This transient presence of the RGEN complex may lead to reduced off-target effects. In contrast, delivery of RGEN components (guide RNA, Cas9 endonuclease) via plasmid DNA sequences can result in constant expression of RGENs from these plasmids which can intensify off target effects (Cradick, T. J. et al (2013) Nucleic Acids Res 41:9584-9592; Fu, Y et al (2014) Nat. Biotechnol. 31:822-826.

Direct delivery can be achieved by combining any one component of the RNA guided endonuclease (guide RNA, Cas protein, mRNA encoding the gRNA or Cas endonuclease) or the RGEN complex itself, with a particle delivery matrix comprising a microparticle such as but not limited to of a gold particle, tungsten particle, and silicon carbide whisker particle. Examples of combination methods described herein for combining microparticles to plasmid DNA and DNA of interest can also be used for coating guide RNA molecules, mRNA molecules, Cas proteins and RGEN complexes to the microparticles.

These coated microparticles can be introduced into the cells by any direct method known in the art such as the particle bombardment method described in Example 8. Microparticles and RGEN components or RGEN complex can be combined (mixed) in any matter to allow for coating of the RGEN components to the micro particles. For example, RGEN components can be precipitated onto gold pellets of a diameter ranging from at least 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm or 1.0 μm in diameter using any suitable buffer (such as but not limiting to a water-soluble cationic lipid such as but not limiting to TransIT-2020 Transfection Reagent (Cat# MIR 5404, Mirus, USA). RGEN component solutions can prepared on ice (or at any temperature suitable to enable mircoparticle bounding) using at least 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 6.0 μg, 7.0 μg, 8.0 μg 9.0 μg or 10 μg of RNA (guided RNA or mRNA) or Cas endonuclease protein. To the pre-mixed RGEN components of RGEN complexes, at least 1 μl to 20 μl of prepared mircoparticles can be added and mixed carefully.

In one embodiment of the disclosure, the method comprises a method of delivering a guide RNA/Cas endonuclease complex into a cell, the method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotide-protein and matrix to bind and form a ribonucleotide-protein—matrix complex; and, introducing said ribonucleotide-protein—matrix complex into said cell. The particle delivery matrix can comprise microparticles combined with a cationic lipid.

The term “cationic lipid” includes reference to a water soluble cationic lipid, such as but not limiting to TransIT-2020, or a cationic lipid solution such as but not limiting to a cationic lipid solution comprising N,N,N′,N′-tetramethyl-N, N′-bis(2-hydroxylethyl)-2,3-di(oleoyloxy)-1,4-butanediammonium iodide, and L-dioleoyl phosphatidylethanolamine (DOPE). (see also US2007/0178593, published on Aug. 2, 2007, incorporated herein by reference),

5. The method of claim.

The particle delivery matrix can comprise microparticles selected from the group consisting of gold particles, tungsten particles, and silicon carbide whisker particles.

The particle delivery matrix can further comprise a compound selected from the group consisting of Tfx-10™, Tfx-20™, Tfx-50™, Lipofectin™, Lipofectamine™, Cellfectin™, Effectene™, Cytofectin GSV™, Perfect Lipids™, DOTAP™, DMRIE-C™, FuGENE-6™, Superfect™, Polyfeet™ polyethyleneimine, chitosan, protamine Cl, histone H1, histone CENH3, poly-L lysine, and DMSA. (US2007/0178593, published on Aug. 2, 2007, incorporated herein by reference)

RGEN components can also be combined prior to be coated on microparticles by combining least 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 6.0 μg, 7.0 μg, 8.0 μg, 9.0 μg or 10 μg of guide RNA with at least 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 6.0 μg, 7.0 μg, 8.0 μg, 9.0 μg or 10 μg of Cas endonuclease in a solution suitable to allow for complex formation (such as but not limiting to a Cas9 buffer (NEB)), at any temperature to allow for complex formation such as a temperature ranging from 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23.0° C., 24° C., 25° C. 26° C. 27° C. 28° C., 29° C., 30° C., 31° C., 32° C., 33.0° C., 34° C. 35° C. 36° C. 37° C. 38° C., 39° C. and 40° C. In one embodiment of the disclosure, the method comprises a method of delivering guide RNA/Cas endonuclease components into a cell, the method comprising introducing at least one guide RNA molecule and at least one Cas endonuclease protein into a cell, and growing said cell under suitable conditions to allow said guide RNA and said Cas endonuclease protein to form a complex inside said cell.

In one embodiment of the disclosure, the method comprises a method of delivering guide RNA/Cas endonuclease components into a cell, the method comprising introducing at least one guide RNA molecule and at least one mRNA encoding a Cas endonuclease protein into a cell, and growing said cell under suitable conditions to allow said mRNA to translate said Cas endonuclease protein and form a complex with said guide RNA

In one embodiment of the disclosure, the method comprises a method of delivering a guide RNA/Cas endonuclease complex into a cell, the method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotide-protein and matrix to bind and form a ribonucleotide-protein—matrix complex; and, introducing said ribonucleotide-protein—matrix complex together with at least one a polynucleotide template into said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of a nucleotide sequence in the genome of said cell, wherein said at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).

In one embodiment of the disclosure, the method comprises a method of delivering a guide RNA/Cas endonuclease complex into a cell, the method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotide-protein and matrix to bind and form a ribonucleotide-protein—matrix complex; and, introducing said ribonucleotide-protein—matrix complex together with a donor DNA into said cell, wherein said donor DNA comprises at least one polynucleotide of interest.

Suitable conditions for growing cells are well known in the art and the skilled artesian can use any growing condition based on the type of cell (such as conditions suitable for plant cells). As described in Example 8, plant embryos or cells can be incubated in any plant maintenance medium known in the art (such as, but not limiting to 560P, Example 8) for 12 to 48 hours at temperatures ranging from 26° C. to 37° C., and then placed at 26° C. After 5 to 7 days the embryos/cells are transferred to any selection medium known in the art (such as, but not limiting to 560R, Example 8), and subcultured thereafter.

RGEN components (including guide RNA, Cas endonuclease protein) can be combined to form a ribonucleotide-protein complex (RNP) prior to be coated on (combined with) microparticles by combining least 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 6.0 μg, 7.0 μg, 8.0 μg, 9.0 μg or 10 μg of guide RNA with at least 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 6.0 μg, 7.0 μg, 8.0 μg, 9.0 μg or 10 μg of Cas endonuclease in a solution suitable to allow for complex formation (such as but not limiting to a Cas9 buffer (NEB)), at any temperature to allow for complex formation such as a temperature ranging from 1° C., 2° C., 3° C., 4° C. 5° C. 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C. 15° C. 16° C. 17° C. 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C. 25° C. 26° C., 27° C. 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C. 35° C. 36° C. 37° C. 38° C., 39° C. and 40° C.

“Mature” protein refers to a post-translationally processed polypeptide (i.e., one from which any pre- or propeptides present in the primary translation product have been removed). “Precursor” protein refers to the primary product of translation of mRNA (i.e., with pre- and propeptides still present). Pre- and propeptides may be but are not limited to intracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, “transient transformation” refers to the transfer of a nucleic acid fragment into the nucleus, or other DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms.

The commercial development of genetically improved germplasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking approach. In this approach, multiple genes conferring different characteristics of interest can be introduced into a plant. Gene stacking can be accomplished by many means including but not limited to co-transformation, retransformation, and crossing lines with different genes of interest.

Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, and plant cells as well as plants and seeds produced by the methods described herein. Plant cells include cells selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tomato, tobacco, Arabidopsis, and safflower cells.

The term “plant” includes reference to whole plants, plant organs, plant tissues, seeds, and plant cells, and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. Plant parts include differentiated and undifferentiated tissues including, but not limited to roots, stems, shoots, leaves, pollens, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos, and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture. The term “plant organ” refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. The term “genome” refers to the entire complement of genetic material (genes and non-coding sequences) that is present in each cell of an organism, or virus or organelle; and/or a complete set of chromosomes inherited as a (haploid) unit from one parent. “Progeny” comprises any subsequent generation of a plant.

A transgenic plant includes, for example, a plant which comprises within its genome a heterologous polynucleotide introduced by a transformation step. The heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. A transgenic plant can also comprise more than one heterologous polynucleotide within its genome. Each heterologous polynucleotide may confer a different trait to the transgenic plant. A heterologous polynucleotide can include a sequence that originates from a foreign species, or, if from the same species, can be substantially modified from its native form. Transgenic can include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The alterations of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods, by the genome editing procedure described herein that does not result in an insertion of a foreign polynucleotide, or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation are not intended to be regarded as transgenic.

In certain embodiments of the disclosure, a fertile plant is a plant that produces viable male and female gametes and is self-fertile. Such a self-fertile plant can produce a progeny plant without the contribution from any other plant of a gamete and the genetic material contained therein. Other embodiments of the disclosure can involve the use of a plant that is not self-fertile because the plant does not produce male gametes, or female gametes, or both, that are viable or otherwise capable of fertilization. As used herein, a “male sterile plant” is a plant that does not produce male gametes that are viable or otherwise capable of fertilization. As used herein, a “female sterile plant” is a plant that does not produce female gametes that are viable or otherwise capable of fertilization. It is recognized that male-sterile and female-sterile plants can be female-fertile and male-fertile, respectively. It is further recognized that a male fertile (but female sterile) plant can produce viable progeny when crossed with a female fertile plant and that a female fertile (but male sterile) plant can produce viable progeny when crossed with a male fertile plant.

Non-conventional yeast herein refers to any yeast that is not a Saccharomyces (e.g., S. cerevisiae) or Schizosaccharomyces yeast species. Non-conventional yeast are described in Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols (K. Wolf, K. D. Breunig, G. Barth, Eds., Springer-Verlag, Berlin, Germany, 2003), which is incorporated herein by reference. Non-conventional yeast in certain embodiments may additionally (or alternatively) be yeast that favor non-homologous end-joining (NHEJ) DNA repair processes over repair processes mediated by homologous recombination (HR). Definition of a non-conventional yeast along these lines—preference of NHEJ over HR—is further disclosed by Chen et al. (PLoS ONE 8:e57952), which is incorporated herein by reference. Preferred non-conventional yeast herein are those of the genus Yarrowia (e.g., Yarrowia lipolytica). The term “yeast” herein refers to fungal species that predominantly exist in unicellular form. Yeast can alternative be referred to as “yeast cells” herein (see also U.S. provisional application 62/036,652, filed on Aug. 13, 2014, which is incorporated by reference herein).

A “centimorgan” (cM) or “map unit” is the distance between two linked genes, markers, target sites, loci, or any pair thereof, wherein 1% of the products of meiosis are recombinant. Thus, a centimorgan is equivalent to a distance equal to a 1% average recombination frequency between the two linked genes, markers, target sites, loci, or any pair thereof.

The present disclosure finds use in the breeding of plants comprising one or more introduced traits. Most commonly, transgenic traits are randomly inserted throughout the plant genome as a consequence of transformation systems based on Agrobacterium, biolistics, or other commonly used procedures. More recently, gene targeting protocols have been developed that enable directed transgene insertion. One important technology, site-specific integration (SSI) enables the targeting of a transgene to the same chromosomal location as a previously inserted transgene. Custom-designed meganucleases and custom-designed zinc finger meganucleases allow researchers to design nucleases to target specific chromosomal locations, and these reagents allow the targeting of transgenes at the chromosomal site cleaved by these nucleases.

The currently used systems for precision genetic engineering of eukaryotic genomes, e.g. plant genomes, rely upon homing endonucleases, meganucleases, zinc finger nucleases, and transcription activator—like effector nucleases (TALENs), which require de novo protein engineering for every new target locus. The highly specific, RNA-directed DNA nuclease, guide RNA/Cas9 endonuclease system described herein, is more easily customizable and therefore more useful when modification of many different target sequences is the goal.

The guide RNA/Cas system described herein is especially useful for genome engineering, especially plant genome engineering, in circumstances where nuclease off-target cutting can be toxic to the targeted cells. In one embodiment of the guide RNA/Cas system described herein, an expression-optimized Cas9 gene, is stably integrated into the target genome, e.g. plant genome. Expression of the Cas9 gene is under control of a promoter, e.g. plant promoter, which can be a constitutive promoter, tissue-specific promoter or inducible promoter, e.g. temperature-inducible, stress-inducible, developmental stage inducible, or chemically inducible promoter. In the absence of the guide RNA or crRNA, the Cas9 protein is not able to cut DNA and therefore its presence in the plant cell should have little or no consequence. Hence a key advantage of the guide RNA/Cas system described herein is the ability to create and maintain a cell line or transgenic organism capable of efficient expression of the Cas9 protein with little or no consequence to cell viability. In order to induce cutting at desired genomic sites to achieve targeted genetic modifications, guide RNAs or crRNAs can be introduced by a variety of methods into cells containing the stably-integrated and expressed cas9 gene. For example, guide RNAs or crRNAs can be chemically or enzymatically synthesized, and introduced into the Cas9 expressing cells via direct delivery methods such a particle bombardment or electroporation. Alternatively, genes capable of efficiently expressing guide RNAs or crRNAs in the target cells can be synthesized chemically, enzymatically or in a biological system, and these genes can be introduced into the Cas9 expressing cells via direct delivery methods such a particle bombardment, electroporation or biological delivery methods such as Agrobacterium mediated DNA delivery.

A guide RNA/Cas system mediating gene targeting can be used in methods for directing transgene insertion and/or for producing complex transgenic trait loci comprising multiple transgenes in a fashion similar as disclosed in WO2013/0198888 (published Aug. 1, 2013) where instead of using a double strand break inducing agent to introduce a gene of interest, a guide RNA/Cas system as disclosed herein is used. A complex trait locus includes a genomic locus that has multiple transgenes genetically linked to each other. By inserting independent transgenes within 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2, or even 5 centimorgans (cM) from each other, the transgenes can be bred as a single genetic locus (see, for example, U.S. patent application Ser. No. 13/427,138) or PCT application PCT/US2012/030061. After selecting a plant comprising a transgene, plants containing (at least) one transgenes can be crossed to form an F1 that contains both transgenes. In progeny from these F1 (F2 or BC1) 1/500 progeny would have the two different transgenes recombined onto the same chromosome. The complex locus can then be bred as single genetic locus with both transgene traits. This process can be repeated to stack as many traits as desired.

Chromosomal intervals that correlate with a phenotype or trait of interest can be identified. A variety of methods well known in the art are available for identifying chromosomal intervals. The boundaries of such chromosomal intervals are drawn to encompass markers that will be linked to the gene controlling the trait of interest. In other words, the chromosomal interval is drawn such that any marker that lies within that interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for northern leaf blight resistance. In one embodiment, the chromosomal interval comprises at least one QTL, and furthermore, may indeed comprise more than one QTL. Close proximity of multiple QTLs in the same interval may obfuscate the correlation of a particular marker with a particular QTL, as one marker may demonstrate linkage to more than one QTL. Conversely, e.g., if two markers in close proximity show co-segregation with the desired phenotypic trait, it is sometimes unclear if each of those markers identifies the same QTL or two different QTL. The term “quantitative trait locus” or “QTL” refers to a region of DNA that is associated with the differential expression of a quantitative phenotypic trait in at least one genetic background, e.g., in at least one breeding population. The region of the QTL encompasses or is closely linked to the gene or genes that affect the trait in question. An “allele of a QTL” can comprise multiple genes or other genetic factors within a contiguous genomic region or linkage group, such as a haplotype. An allele of a QTL can denote a haplotype within a specified window wherein said window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers. A haplotype can be defined by the unique fingerprint of alleles at each marker within the specified window.

A variety of methods are available to identify those cells having an altered genome at or near a target site without using a screenable marker phenotype. Such methods can be viewed as directly analyzing a target sequence to detect any change in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blots, and any combination thereof.

Proteins may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of the protein(s) can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel et al., (1987) Meth Enzymol 154:367-82; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance regarding amino acid substitutions not likely to affect biological activity of the protein is found, for example, in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, D.C.). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable. Conservative deletions, insertions, and amino acid substitutions are not expected to produce radical changes in the characteristics of the protein, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by routine screening assays. Assays for double-strand-break-inducing activity are known and generally measure the overall activity and specificity of the agent on DNA substrates containing target sites.

The term “dicot” refers to the subclass of angiosperm plants also knows as “dicotyledoneae” and includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of the same. Plant cell, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

The term “crossed” or “cross” or “crossing” in the context of this disclosure means the fusion of gametes via pollination to produce progeny (i.e., cells, seeds, or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, i.e., when the pollen and ovule (or microspores and megaspores) are from the same plant or genetically identical plants).

The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny plant via a sexual cross between two parent plants, where at least one of the parent plants has the desired allele within its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., a transgene, a modified (mutated or edited) native allele, or a selected allele of a marker or QTL.

Standard DNA isolation, purification, molecular cloning, vector construction, and verification/characterization methods are well established, see, for example Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, N.Y.). Vectors and constructs include circular plasmids, and linear polynucleotides, comprising a polynucleotide of interest and optionally other components including linkers, adapters, regulatory or analysis. In some examples a recognition site and/or target site can be contained within an intron, coding sequence, 5′ UTRs, 3′ UTRs, and/or regulatory regions.

The present disclosure further provides expression constructs for expressing in a plant, plant cell, or plant part a guide RNA/Cas system that is capable of binding to and creating a double strand break in a target site. In one embodiment, the expression constructs of the disclosure comprise a promoter operably linked to a nucleotide sequence encoding a Cas gene and a promoter operably linked to a guide RNA of the present disclosure. The promoter is capable of driving expression of an operably linked nucleotide sequence in a plant cell.

Any plant can be used, including monocot and dicot plants. Examples of monocot plants that can be used include, but are not limited to, corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), wheat (Triticum aestivum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), banana (Musa spp.), palm, ornamentals, turfgrasses, and other grasses. Examples of dicot plants that can be used include, but are not limited to, soybean (Glycine max), canola (Brassica napus and B. campestris), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum), and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), potato (Solanum tuberosum) etc.

The meaning of abbreviations is as follows: “sec” means second(s), “min” means minute(s), “h” means hour(s), “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” means micromolar, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” means microgram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means base pair(s) and “kb” means kilobase(s).

Non-limiting examples of compositions and methods disclosed herein are as follows: 1. A method for restoring function to a non-functional gene product in the genome of a cell, the method comprising introducing a guide RNA/Cas endonuclease complex into a cell comprising a disrupted gene in its genome, wherein said complex creates a double strand break, wherein said disrupted gene does not encode a functional gene product, wherein said disrupted gene is restored without the use of a polynucleotide modification template to a non-disrupted gene capable of encoding said functional gene product. 2. The method of embodiment 1, wherein said disrupted gene comprises a base pair deletion of the 4th nucleotide upstream (5′) of a PAM sequence when compared to its corresponding non-disrupted gene, wherein said base pair deletion creates an amino acid frameshift in the gene product of the disrupted gene thereby rendering the gene product of the disrupted gene non-functional. 3. The method of embodiment 2, wherein the base pair deletion is the first nucleotide of a codon sequence. 4. The method of embodiment 2, wherein the base pair deletion is the second nucleotide of a codon sequence. 5. The method of embodiment 2, wherein the base pair deletion is the third nucleotide of a codon sequence. 6. The method of embodiment 1, wherein the restoration is accomplished by Non-Homologous-End-Joining (NHEJ) resulting in the insertion of a single base at the double strand break site. 7. The method of embodiment 1, wherein the restoration is accomplished by the insertion of a single base at the double strand break site without the use of Homologous Recombination or homology-directed repair. 8. A method for modifying a nucleotide sequence in the genome of a cell, the method comprising:

introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and,

selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable protein.

9. The method of embodiment 8, wherein the modification is selected from the group consisting of an insertion of at least one nucleotide, a deletion of at least one nucleotide, or a substitution of at least one nucleotide in said target site. 10. The method of embodiment 8, further comprising introducing a polynucleotide modification template into said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence. 11. The method of embodiment 8, wherein the at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). 12. The method of embodiment 8, further comprising introducing a donor DNA into the cell of (a) wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site. 13. The method of embodiment 8, wherein the cell is selected from the group consisting of a human, non-human, animal, archaea, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cell. 14. The method of embodiment 13, wherein the plant cell is selected from the group consisting of a monocot and dicot cell. 15. The method of embodiment 14, wherein the plant cell is selected from the group consisting of a maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tomato, tobacco, Arabidopsis, and safflower cell. 16. The method of embodiment 13, further comprising producing a plant or progeny plant from said plant cell. 17. A plant or progeny plant produced by the method of embodiment 16, wherein said plant or progeny plant is void of any one guide RNA and Cas endonucleases. 18. The method of embodiment 8, wherein the disrupted selectable marker gene is a disrupted ALS-resistance gene. 19. A method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising:

-   -   a) introducing into at least one cell at least one guide RNA and         at least one Cas endonuclease, wherein said guide RNA and Cas         endonuclease can form a complex capable of introducing a double         strand break in said nucleotide sequence;     -   b) selecting a cell from (a) comprising at least one single         nucleotide deletion in said nucleotide sequence, wherein said         nucleotide deletion is located at a position to be edited; and,     -   c) introducing into a cell of (b) at least one guide RNA and at         least one Cas endonuclease, wherein said guide RNA and Cas         endonuclease can form a complex capable of introducing a double         strand break in said nucleotide sequence and insert a single         nucleotide at the same position of the nucleotide deletion         of (b) without the use of a polynucleotide modification         template.         20. A method for editing a nucleotide sequence in the genome of         a plant without the use of a polynucleotide modification         template or donor DNA, the method comprising:     -   a) introducing into at least one plant cell at least one guide         RNA and at least one Cas endonuclease, wherein said guide RNA         and Cas endonuclease can form a complex capable of introducing a         double strand break in said nucleotide sequence;     -   b) selecting a plant cell from (a) comprising at least one         single nucleotide deletion in said nucleotide sequence, wherein         said nucleotide deletion is located at a position to be edited;     -   c) regenerating a plant from the plant cell of (b);     -   d) introducing into a cell from the plant of (c) at least one         guide RNA and at least one Cas endonuclease, wherein said guide         RNA and Cas endonuclease can form a complex capable of         introducing a double strand break in said nucleotide sequence         and inserting a single nucleotide at the same position of the         nucleotide deletion of (b) without the use of a polynucleotide         modification template; and,     -   e) optimally, selecting a cell comprising the nucleotide         insertion of (d).         21. The method of any one of embodiments 1, 8, 18-20, wherein         the guide RNA and Cas endonuclease protein forming the guide         RNA/Cas endonuclease complex are introduced into the cell as RNA         and protein, respectively.         22. The method of any one of embodiments 1, 8, 18-20, wherein         the guide RNA/Cas endonuclease complex is assembled in vitro         prior to being introduced intyo the plant cell, and introduced         into the cell as a ribonucleotide-protein complex.         23. The method of any one of embodiments 1, 8, 18-20 wherein         components of the guide RNA/Cas endonuclease complex are         introduced as mRNAencoding the Cas endonuclease protein and as         RNA comprising the guide RNA.         24. The method of any one of embodiments 1, 8, 18-19 wherein         components of the guide RNA/Cas endonuclease complex are         introduced as recombinant DNA molecules encoding the guide         molecule and the Cas endonuclease protein.         25. The method of any one of embodiments 1, 8, 18-19 wherein the         guide RNA/Cas endonuclease complex is assembled inside the cell.         26. A method of delivering a guide RNA/Cas endonuclease complex         into a cell, the method comprising combining at least one guide         RNA molecule and at least one Cas endonuclease protein to form a         ribonucleotide-protein and combining said ribonucleotide-protein         with a particle delivery matrix to allow for said         ribonucleotide-protein and matrix to bind and form a         ribonucleotide-protein—matrix complex; and, introducing said         ribonucleotide-protein—matrix complex into said cell.         27. A method of delivering guide RNA/Cas endonuclease components         into a cell, the method comprising introducing at least one         guide RNA molecule and at least one Cas endonuclease protein         into a cell, and growing said cell under suitable conditions to         allow said guide RNA and said Cas endonuclease protein to form a         complex inside said cell.         28. A method of delivering guide RNA/Cas endonuclease components         into a cell, the method comprising introducing at least one         guide RNA molecule and at least one mRNA encoding a Cas         endonuclease protein into a cell, and growing said cell under         suitable conditions to allow said mRNA to translate said Cas         endonuclease protein and form a complex with said guide RNA.         29. The method of embodiments 26-28, further comprising         introducing a polynucleotide template, wherein said         polynucleotide modification template comprises at least one         nucleotide modification of a nucleotide sequence in the genome         of said cell, wherein said at least one nucleotide modification         of said polynucleotide modification template is selected from         the group consisting of (i) a replacement of at least one         nucleotide, (ii) a deletion of at least one nucleotide, (iii) an         insertion of at least one nucleotide, and (iv) any combination         of (i)-(iii).         30. The method of embodiments 26-28, further comprising         introducing a donor DNA, wherein said donor DNA comprises at         least one polynucleotide of interest.         31. The method of embodiments 26-28, wherein said guide RNA/Cas         endonuclease complex introduces a double strand break at a         target site in the genome of said cell.         32. The method of embodiments 1-31, wherein said Cas         endonuclease is selected from the group consisting of a Cas9         protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3         protein, Cas3, Cas3-H, Cas 5, Cas7, Cas8, Cas10, or complexes of         these.         33. The method of embodiments 26-28, wherein the introducing is         via a delivery system selected from the group consisting of         particle mediated delivery, whisker mediated delivery,         cell-penetrating peptide mediated delivery, electroporation,         PEP-mediated transfection and nanoparticle mediated delivery.         34. The method of embodiment 29, wherein the polynucleotide         modification template is a single stranded or double stranded         molecule.         35. The method of embodiment 30, wherein the donor DNA is a         single stranded or double stranded molecule.         36. The method of embodiments 26-28, wherein the cell is a plant         cell that comprises pre-integrated developmental genes capable         of stimulating cell development.         37. The method of embodiments 26-28, wherein the cell is         selected from the group consisting of a human, non-human,         animal, archaea, bacterial, fungal, insect, yeast,         non-conventional yeast, and plant cell.         38. The method of embodiment 37, wherein the plant cell is         selected from the group consisting of a monocot and dicot cell.         39. The method of embodiment 38, wherein the plant cell is         selected from the group consisting of a maize, rice, sorghum,         rye, barley, wheat, millet, oats, sugarcane, turfgrass, or         switchgrass, soybean, canola, alfalfa, sunflower, cotton,         tobacco, peanut, potato, tomato, tobacco, Arabidopsis, and         safflower cell.         40. A plant produced from the plant cell of embodiment 36,         wherein said plant comprises said at least one nucleotide         modification in the genome of said plant cell and wherein said         plant does not comprise said guide RNA/Cas endonuclease complex         or any component thereof.         41. A plant produced from the plant cell of embodiment 37,         wherein said plant comprises at least one polynucleotide of         interest integrated into its genome.         42. The method of embodiment 26, wherein said particle delivery         matrix comprises a microparticle.         43. The method of embodiment 42, wherein said microparticle is         selected from the group consisting of a gold particle, a         tungsten particle, and a silicon carbide whisker particle.         44. The method of embodiment 26, wherein the particle delivery         matrix comprises microparticles combined with a cationic lipid.         45. The method of embodiment 44, wherein the cationic lipid is a         water soluble cationic lipid.         46. The method of embodiment 45, wherein the water soluble         cationic lipid is TransIT-2020.         47. A method for producing a sulfonylurea resistant plant         comprising a modified target site, the method comprising: a)         introducing to a plant cell comprising a disrupted sulfonylurea         resistant (ALS) gene, a first guide RNA, a Cas9 endonuclease, at         least a second guide RNA, wherein said first guide RNA and Cas9         endonuclease can form a first complex capable of introducing a         double strand break immediately downstream (3′) of a second         nucleotide of a codon sequence located in said disrupted         sulfonylurea resistant (ALS) gene, wherein said second guide RNA         and Cas9 endonuclease can form a second complex capable of         introducing a double strand break at said target site; and, b)         obtaining a sulfonylurea resistant plant from said plant cell,         wherein said sulfonylurea resistant plant comprises a         modification at said target, wherein said modification is         selected from the group consisting of (i) a replacement of at         least one nucleotide, (ii) a deletion of at least one         nucleotide, (iii) an insertion of at least one nucleotide,         and (iv) any combination of (i)-(iii).         48. A method for restoring function to a non-functional gene         product in the genome of a cell, the method comprising         introducing a guide polynucleotide/Cas endonuclease complex into         a cell comprising a disrupted gene in its genome, wherein said         complex creates a double strand break, wherein said disrupted         gene does not encode a functional gene product, wherein said         disrupted gene is restored without the use of a polynucleotide         modification template to a non-disrupted gene capable of         encoding said functional gene product.         49. The method of embodiment 46, wherein said disrupted gene         comprises a base pair deletion of the 4^(th) nucleotide upstream         (5′) of a PAM sequence when compared to its corresponding         non-disrupted gene, wherein said base pair deletion creates an         amino acid frameshift in the gene product of the disrupted gene         thereby rendering the gene product of the disrupted gene         non-functional.         48. A method for modifying a nucleotide sequence in the genome         of a cell, the method comprising:

introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide polynucleotide, a Cas endonuclease, and at least a second guide polynucleotide, wherein said first guide polynucleotide and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said second guide polynucleotide and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and,

selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein.

50. A method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising:

-   -   a) introducing into at least one cell at least one guide         polynucleotide and at least one Cas endonuclease, wherein said         guide polynucleotide and Cas endonuclease can form a complex         capable of introducing a double strand break in said nucleotide         sequence;     -   b) selecting a cell from (a) comprising at least one single         nucleotide deletion in said nucleotide sequence, wherein said         nucleotide deletion is located at a position to be edited; and,     -   c) introducing into a cell of (b) at least one guide         polynucleotide and at least one Cas endonuclease, wherein said         guide polynucleotide and Cas endonuclease can form a complex         capable of introducing a double strand break in said nucleotide         sequence and insert a single nucleotide at the same position of         the nucleotide deletion of (b) without the use of a         polynucleotide modification template.         51. A method of delivering a guide polynucleotide/Cas         endonuclease complex into a cell, the method comprising         combining at least one guide polynucleotide molecule and at         least one Cas endonuclease protein to form a         ribonucleotide-protein and combining said ribonucleotide-protein         with a particle delivery matrix to allow for said         ribonucleotide-protein and matrix to bind and form a         ribonucleotide-protein—matrix complex; and, introducing said         ribonucleotide-protein—matrix complex into said cell.

EXAMPLES

In the following Examples, unless otherwise stated, parts and percentages are by weight and degrees are Celsius. It should be understood that these Examples, while indicating embodiments of the disclosure, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Such modifications are also intended to fall within the scope of the appended claims.

Example 1 Modifying Target DNA Sequences in the Genome of a Plant Cell by Delivering Cas9 Endonuclease and Guide RNA Expression Cassettes

The Cas9 gene from Streptococcus pyogenes M1 GAS (SF370) (SEQ ID NO: 1) was maize codon optimized using standard techniques known in the art and the potato ST-LS1 intron (SEQ ID NO: 2) was introduced in order to eliminate its expression in E. coli and Agrobacterium. To facilitate nuclear localization of the Cas9 protein in maize cells, Simian virus 40 (SV40) monopartite amino terminal nuclear localization signal (MAPKKKRKV, SEQ ID NO: 3) and Agrobacterium tumefaciens bipartite VirD2 T-DNA border endonuclease carboxyl terminal nuclear localization signal (KRPRDRHDGELGGRKRAR, SEQ ID NO: 4) were incorporated at the amino and carboxyl-termini of the Cas9 open reading frame, respectively. The maize optimized Cas9 gene was operably linked to a maize constitutive promoter (Ubiquitin) by standard molecular biology techniques. Transcription is terminated by the addition of the 3′ sequences from the potato proteinase inhibitor II gene (PinII) to generate UBI:Cas9:PinII vector. The sequence of the Ubiquitin driven maize optimized Cas9 expression cassette is shown in SEQ ID NO: 5.

Single guide RNAs (gRNAs) were designed using the methods described by Mali et al., 2013 (Science 339:823-26). A maize U6 polymerase III promoter and terminator were isolated and used to direct initiation and termination of gRNAs, respectively. Two BbsI restriction endonuclease sites were introduced in an inverted tandem orientation with cleavage orientated in an outward direction as described in Cong et al., 2013 (Science 339:819-23) to facilitate the rapid introduction of maize genomic DNA target sequences into the gRNA expression constructs. Only target sequences starting with a G nucleotide were used to promote favorable polymerase III expression of the gRNA. The gRNA expression cassettes were subcloned into Bluescript SK vector (SEQ ID NO: 6).

To test whether the maize optimized Cas9-gRNA complex could recognize, cleave, and facilitate targeted mutations in maize chromosomal DNA through non-homologous end joining (NHEJ) repair pathway, 5 maize loci (three different genomic sequences in each locus) were targeted for cleavage (see Table 2) and examined by amplicon deep sequencing for the presence of mutations.

TABLE 2  Maize genomic sites targeted by the  Cas9-gRNA system Target Site  Maize Genomic SEQ Desig- Target Site  PAM ID Locus Location nation Sequence Sequence NO: M526 Chr. 1: MS26Cas-1 GTACTCCATCCGC GGG 7 51.81cM CCCATCGAGTA MS26Cas-2 GCACGTACGTCAC CGG 8 CATCCCGC MS26Cas-3 GACGTACGTGCCC GGG 9 TACTCGAT LIG Chr. 2: LIGCas-1 GTACCGTACGTGC AGG 10 28.45cM CCCGGCGG LIGCas-2 GGAATTGTACCGT CGG 11 ACGTGCCC LIGCas-3 GCGTACGCGTACG AGG 12 TGTG MS45 Chr. 9: MS45Cas-1 GCTGGCCGAGGTC CGG 13 119.15cM GACTAC MS45Cas-2 GGCCGAGGTCGAC CGG 14 TACCGGC MS45Cas-3 GGCGCGAGCTCGT CGG 15 GCTTCAC ALS1 1-Chr. 4: ALSCas-1 GGTGCCAATCATG CGG 16 and 107.73cM CGTCG ALS2 2-Chr. 5: ALSCas-2 GGTCGCCATCACG AGG 17 115.49cM GGAC ALSCas-3 GTCGCGGCACCTG TGG 18 TCCCGTGA MS26 = Male Sterility Gene 26, LIG = Liguleless-1 Gene Promoter, MS45 = Male Sterility Gene 45, ALS1 = Acetolactate Synthase Gene 1 (Chr. 4), ALS1 = Acetolactate Synthase Gene 2 (Chr. 5).

The maize optimized Cas9 endonuclease and gRNA expression cassettes containing the specific maize variable targeting domains were co-delivered to 60-90 Hi-II immature maize embryos by particle bombardment (see Example 8) with selectable and visible marker (UBI:MoPAT:DsRED fusion) and developmental genes ZmODP-2 (BBM) and ZmWUS2 (WUS) (see Example 9). Hi-II maize embryos transformed with only the Cas9 or gRNA expression cassette served as negative controls. After 7 days, 20-30 most uniformly transformed embryos from each treatment (based on transient expression of the DsRED fluorescent protein) were pooled and total genomic DNA was extracted. The region surrounding the intended target site was PCR amplified with Phusion® High Fidelity PCR Master Mix (New England Biolabs, M0531 L) adding sequences necessary for amplicon-specific barcodes and Illumnia sequencing using “tailed” primers through two rounds of PCR. The primers used in the primary PCR reaction are shown in Table 3.

TABLE 3  PCR primer sequences SEQ Target ID Site Primer Primary PCR Primer Sequence NO: MS26Cas-1 Forward CTACACTCTTTCCCTACACGACGCTCTTCC 19 GATCTAGGACCGGAAGCTCGCCGCGT MS26Cas-1 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 20 ATCTTCCTGGAGGACGACGTGCTG MS26Cas-2 Forward CTACACTCTTTCCCTACACGACGCTCTTCC 21 GATCTAAGGTCCTGGAGGACGACGTGCTG MS26Cas-2 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 22 ATCTCCGGAAGCTCGCCGCGT MS26Cas-3 Forward CTACACTCTTTCCCTACACGACGCTCTTCC GATCTTCCTCCGGAAGCTCGCCGCGT 23 MS26Cas-3 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 20 ATCTTCCTGGAGGACGACGTGCTG LIGCas-1 Forward CTACACTCTTTCCCTACACGACGCTCTTCC 24 GATCTAGGACTGTAACGATTTACGCACCTG CTG LIGCas-1 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 25 ATCTGCAAATGAGTAGCAGCGCACGTAT LIGCas-2 Forward CTACACTCTTTCCCTACACGACGCTCTTCC 26 GATCTTCCTCTGTAACGATTTACGCACCTG CTG LIGCas-2 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 25 ATCTGCAAATGAGTAGCAGCGCACGTAT LIGCas-3 Forward CTACACTCTTTCCCTACACGACGCTCTTCC 27 GATCTAAGGCGCAAATGAGTAGCAGCGCAC LIGCas-3 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 28 ATCTCACCTGCTGGGAATTGTACCGTA MS45Cas-1 Forward CTACACTCTTTCCCTACACGACGCTCTTCC 29 GATCTAGGAGGACCCGTTCGGCCTCAGT MS45Cas-1 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 30 ATCTGCCGGCTGGCATTGTCTCTG MS45Cas-2 Forward CTACACTCTTTCCCTACACGACGCTCTTCC 31 GATCTTCCTGGACCCGTTCGGCCTCAGT MS45Cas-2 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 30 ATCTGCCGGCTGGCATTGTCTCTG MS45Cas-3 Forward CTACACTCTTTCCCTACACGACGCTCTTCC 32 GATCTGAAGGGACCCGTTCGGCCTCAGT MS45Cas-3 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 30 ATCTGCCGGCTGGCATTGTCTCTG ALSCas-1 Forward CTACACTCTTTCCCTACACGACGCTCTTCC GATCTAAGGCGACGATGGGCGTCTCCTG 33 ALSCas-1 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 34 ATCTGCGTCTGCATCGCCACCTC ALSCas-2 Forward CTACACTCTTTCCCTACACGACGCTCTTCC GATCTTTCCCGACGATGGGCGTCTCCTG 35 ALSCas-2 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 34 ATCTGCGTCTGCATCGCCACCTC ALSCas-3 Forward CTACACTCTTTCCCTACACGACGCTCTTCC GATCTGGAACGACGATGGGCGTCTCCTG 36 ALSCas-3 Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCG 34 ATCTGCGTCTGCATCGCCACCTC

Primers used in the secondary PCR reaction were AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACG (forward, SEQ ID NO: 37) and CAAGCAGAAGACGGCATA (reverse, SEQ ID NO: 38).

The resulting PCR amplifications were purified with a Qiagen PCR purification spin column, concentration measured with a Hoechst dye-based fluorometric assay, combined in an equimolar ratio, and single read 100 nucleotide-length deep sequencing was performed on Illumina's MiSeq Personal Sequencer with a 30-40% (v/v) spike of PhiX control v3 (Illumina, FC-110-3001) to off-set sequence bias. Only those reads with a ≥1 nucleotide indel arising within the 10 nucleotide window centered over the expected site of cleavage and not found in a similar level in the negative control were classified as mutations. Mutant reads with the same mutation were counted and collapsed into a single group and the top 10 most prevalent mutations were visually confirmed as arising within the expected site of cleavage. The total numbers of mutations were then used to calculate the percentage of mutant reads based on the total number of reads of an appropriate length containing a perfect match to the barcode and forward primer.

The mutation frequencies revealed by amplicon deep sequencing for the Cas9-gRNA system targeting all 15 sites are shown in Table 4.

TABLE 4 Percent of mutant reads at 5 target loci (15 target sites) Number of Percentage Total Mutant of Mutant Number of Target Reads in Target DSB Reagents Reads Gene Reads Target Gene LIG LIG-CR1 716,854 33,050 4.61% (Chr. 2) gRNA + Cas9 LIG-CR2 711,047 16,675 2.35% gRNA + Cas9 LIG-CR3 713,183 27,959 3.92% gRNA + Cas9 MS26 MS26-CR1 575,671 10,073 1.75% (Chr. 1) gRNA + Cas9 MS26-CR2 543,856 16,930 3.11% gRNA + Cas9 MS26-CR3 538,141 13,879 2.58% gRNA + Cas9 MS45 MS45-CR1 812,644 3,795 0.47% (Chr. 9) gRNA + Cas9 MS45-CR2 785,183 14,704 1.87% gRNA + Cas9 MS45-CR3 728,023 9,203 1.26% gRNA + Cas9 ALS1 ALS-CR1 434,452 9,669 2.23% (Chr. 4) gRNA + Cas9 and ALS-CR2 472,351 6,352 1.35% ALS2 gRNA + Cas9 (Chr. 5) ALS-CR3 497,786 8,535 1.72% gRNA + Cas9 Controls Cas9 only 640,063 1 0.00% LIG-CR1 gRNA 646,774 1 0.00% only

Further analysis demonstrated, that the most common type of mutations promoted by Cas9-gRNA system was single nucleotide insertions (for example, see FIG. 1, SEQ ID NOs: 49-58). Similar results were observed for the majority of gRNAs tested (Table 5).

TABLE 5 Frequency of a single nucleotide insertions and deletions in 15 target sites promoted by the Cas9-gRNA system % Single nt Insertion % Single nt Deletion Target of Total Number of Total Number Locus DSB Reagents of Mutant Reads of Mutant Reads LIG LIG-CR1 86%  5% gRNA + Cas9 LIG-CR2 49% 25% gRNA + Cas9 LIG-CR3 62% 20% gRNA + Cas9 MS26 MS26-CR1 46% 16% gRNA + Cas9 MS26-CR2 78%  8% gRNA + Cas9 MS26-CR3 45% 18% gRNA + Cas9 MS45 MS45-CR1 45% 17% gRNA + Cas9 MS45-CR2 41% 23% gRNA + Cas9 MS45-CR3 20% 24% gRNA + Cas9 ALS1 ALS-CR1 22% 76% (Chr. 4) gRNA + Cas9 and ALS-CR2 60% 27% ALS2 gRNA + Cas9 (Chr. 5) ALS-CR3 84% 12% gRNA + Cas9

This example demonstrates that RNA guided Cas9 generates double strand breaks resulting in high frequency of mutations. Analysis of mutations in multiple target sites showed that although various size deletions and/or insertions were observed, a single nucleotide insertion and a single nucleotide deletion were the most prevalent types of mutations generated by the Cas9-gRNA technology for the majority of the target sites tested in maize.

Example 2 Edited Acetolactate Synthase Gene Confers Resistance to Chlorsulfuron

This example demonstrates that specific change(s) introduced into the nucleotide sequence of the native maize acetolactate synthase (ALS) gene result in resistance to sulfonylurea class herbicides, specifically, chlorsulfuron.

There are two ALS genes in maze, ALS1 (SEQ ID NO: 39) and ALS2 (SEQ ID NO: 40), located on chromosomes 4 and 5, respectively, with 94% sequence identity at the DNA level.

The ALS protein contains N-terminal transit and the mature protein is formed following transport into the chloroplast and subsequent cleavage of the transit peptide. The mature protein starts at residue S41, resulting in a mature protein of 598 amino acids with a predicted molecular weight of 65 kDa (SEQ ID NO: 41).

Modification of a nucleotide sequence of either ALS1 or ALS2 resulting in a single amino acid residue (P165A or P165S, boxed in grey) change in comparison to the endogenous maize acetolactate synthase protein provides resistance to herbicides in maize.

As acetolactate synthase is a critical enzyme for cell function in plants, simultaneous bi-allelic knockouts of ALS1 and ALS2 genes would not be expected to survive. Therefore, based on polymorphism between ALS1 and ALS2 nucleotide sequences, ALS2-specific ALSCas-4 target site was identified and tested. ALSCas-1 guide RNA expressing construct targeting both ALS1 and ALS2 genes was used as control. Table 6 presents information about ALSCas-1 and ALSCas-4 target sites.

TABLE 6  ALSCas-1 and ALSCas-4 (ALS2-specific) target sites Maize Genomic  SEQ Target Site Target Site PAM ID Loci Location Designation Sequence Sequence NO: ALS1 Chr. 4: ALSCas-1 GGTGCCAATCATG CGG 16 and 107.73c CGTCG ALS2 M and GCTGCTCGATTCC Chr. 5: ALSCas-4 GTCCCCA TGG 42 115.49c M Underlined nucleotides in the ALSCas-4 target site and PAM are different in the ALS1 gene.

Mutation frequencies at the ALSCas-1 and ALSCas-4 were determined by amplicon deep sequencing as described in Example 1 and shown in Table 7.

TABLE 7 Frequencies of mutations at ALSCas-1 and ALSCas-4 target sites recovered by amplicon deep sequencing. Mutant reads Mutant reads Target Site Total Reads (ALS1) (ALS2) ALSCas-1 204,230 2704 (1.3%) 5072 (2.5%) ALSCas-4 120,766   40 (0.03%) 3294 (2.7%)

These results demonstrated that ALSCas-4 gRNA/Cas9 system mutated the ALS2 gene with approximately 90 times higher efficiency than the ALS1 gene. Therefore, the ALSCas-4 target site and the corresponding ALS-CR4 gRNA were selected for the ALS gene editing experiment.

To generate ALS2 edited alleles, a 794 bp polynucleotide modification template comprising a fragment of homology (SEQ ID NO: 43) was cloned into a plasmid vector and two 127 nt single-stranded polynucleotide modification templates (also referred to as DNA oligos, Oligo1, SEQ ID NO: 44, and Oligo2, SEQ ID NO: 45) were tested as polynucleotide modification templates (FIG. 2). The 794 bp fragment had the same sequence modifications as Oligo1. The polynucleotide modification templates (repair templates) contained several nucleotide changes in comparison to the native sequence. Single-stranded Oligo1 and the 794 bp repair templates included a single nucleotide change which would direct editing of DNA sequences corresponding to the proline at amino acid position 165 to a serine (P165S), as well as three additional changes within the ALS-CR4 target site and PAM sequence. Modification of the PAM sequence within the repair template altered the methionine codon (AUG) to isoleucine (AUU), which naturally occurs in the ALS1 gene. A second 127 nt single-stranded oligo repair template (Oligo2) was also tested which preserved the methionine at position 157 but contained three additional single nucleotide changes in the sequence which would influence base pairing with the ALS-CR4 gRNA (FIG. 2).

Approximately 1,000 immature embryos per treatment were bombarded with the two oligo or single plasmid repair templates, Cas9, ALS-CR4 gRNA, and MoPAT-DsRED in DNA expression cassettes and placed on media to select for bialaphos resistance conferred by PAT. Five weeks post-transformation, two hundred (per treatment) randomly selected independent young callus sectors growing on selective media were separated from the embryos and transferred to fresh bialaphos plates. The remaining embryos (>800 per treatment) with developing callus events were transferred to the plates containing 100 ppm of chlorosulfuron as direct selection for an edited ALS2 gene. A month later, a total of 384 randomly picked callus sectors growing on bialaphos (approximately 130 events for each repair template) and 7 callus sectors that continued growing on media with chlorsulfuron were analyzed by PCR amplification and sequencing. Edited ALS2 alleles were detected in nine callus sectors: two derived from the callus sectors growing on bialaphos and generated using the 794 bp repair DNA template, and the remaining 7 derived from chlorosulfuron resistant callus sectors edited using the 127 nt single-stranded oligos, three by Oligo1 and four by Oligo2. The second ALS2 allele in these callus sectors was mutated as a result of NHEJ repair. Analysis of the ALS1 gene revealed only wild-type sequence confirming high specificity of the ALS-CR4 gRNA.

Plants were regenerated from 7 out of 9 callus sectors containing edited ALS2 alleles for additional molecular analysis and progeny testing. DNA sequence analysis of ALS2 alleles confirmed the presence of the P165S modifications (ALS2-P165S) as well as the other nucleotide changes associated with the respective repair templates. T1 and T2 progeny of two TO plants generated from different callus events (794 bp repair DNA and Oligo2) were analyzed to evaluate the inheritance of the edited ALS2 alleles. Progeny plants derived from crosses using pollen from wild type Hi-II plants were analyzed by sequencing and demonstrated sexual transmission of the edited alleles observed in the parent plant with expected 1:1 segregation ratio (57:56 and 47:49, respectively). To test whether the edited ALS sequence confers herbicide resistance, selected four-week old segregating T1 plants with edited and wild-type ALS2 alleles were sprayed with four different concentrations of chlorsulfuron (50, 100 (1×), 200, and 400 mg/liter). Three weeks after treatment, plants with an edited allele showed normal phenotype (FIG. 3—left), while plants with only wild-type alleles demonstrated strong signs of senescence (FIG. 3—right).

In addition to resistance to sulfonylurea class herbicides (specifically, chlorsulfuron), ALS genes can be modified to confer resistance to other classes of AHAS inhibitors including triazolopy-rimidines, pyrimidinylthio-benzoates, and Imidazolinone herbicides (Tan S, Evans R R, Dahmer M L, Singh B K, Shaner D L (2005) Imidazolinone-tolerant crops: history, current status and future. Pest Management Science 61: 246-257). Thus, modifications to ALS genes should not be limited to changes describe herein and conferring chlorsulfuron resistance.

These experiments demonstrate that Cas9-gRNA can stimulate HDR-dependent targeted sequence modifications in maize resulting in plants with an edited endogenous gene which properly transmits to subsequent generations. The data also indicate that a single edited ALS2 allele under endogenous promoter provides herbicide resistance in maize.

Example 3 ALS2 as Endogenous Selectable Marker Gene

This example demonstrates how specifically edited ALS2 gene can be used to generate a selectable marker in a cell replacing delivery of exogenous marker genes currently used in plant transformation.

Due to the relatively low frequencies of plant transformation (transgenic event recovery), selectable marker genes providing resistance to various herbicides are routinely co-delivered with trait genes. To confer resistance, these selectable marker genes need to be stably integrated into the plant genome and have to be excised or bred out in consecutive generations. Some native plant genes can be specifically modified (edited) to confer resistance to herbicides. As described in Example 2, the ALS2 gene with a single amino acid change provides resistance to chlorsulfuron. Therefore, it may be anticipated that gene mutagenesis, gene editing or co-delivery of a trait gene and coincident ALS2 gene editing can be used without an exogenously supplied marker gene. Giving high mutation frequency as the result of NHEJ repair of DSB generated by Cas9-gRNA system (Example 1), this approach might be useful for gene mutagenesis. In this case, the frequency of mutated events would be anticipated to be dependent on HDR-mediated ALS gene editing. With respect to gene editing, it is likely that the combination of two low frequency HDR-dependent genome editing processes (one for ALS gene repair for selection and another for endogenous gene editing or trait gene integration) in plant cells would make the approach using coincident ALS2 gene editing rather impractical.

The following example describes a method that allows overcoming this low efficiency (and impracticality) and improving the likelihood of selecting for plant cells resistant to selective agents. The method does not rely on HDR-dependent gene editing but rather relies on the restoration of the gene function by targeted mutagenesis through NHEJ DNA repair, which is more common (than HDR) in plant somatic cells. As described in Example 2, there are two ALS genes in maize, ALS1 and ALS2, located on chromosomes 4 and 5, respectively. Specific editing of either one of the two ALS genes will confer herbicide resistance. These genes play an essential role in plant metabolism, consequently, targeting and mutating both of them at the same time leads to the cell death. Therefore, in this example, modifications only involve the ALS2 gene by using an ALS2-specific gRNA that does not target the ALS1 gene, hence ALS1 remains wild type. Specifically, two modifications are introduced into the ALS2 gene; first, specific nucleotide(s) change, for example, C to T at the nucleotide position 493 (FIG. 2, oligo1) or C to T and C to G at the nucleotide positions 493 and 495, respectively (FIG. 2, oligo2) to convert Proline to Serine at amino acid position 165 (named ALS2-P165S) conferring resistance to chlorsulfuron (see Example 2 for details). Second, removal of a single nucleotide, for example a G at the nucleotide position 165 (FIG. 4A-4B) resulting in the translational frameshift (FIG. 4B) and, hence, loss of ALS2-mediated chlorsulfuron resistance (named ALS2-P165S-CCΔ). While many designs are anticipated, for the highest frequency of ALS2 gene repair, the single nucleotide position (example shown in FIG. 4A-4C) should preferably be: i) the 4^(th) nucleotide upstream (5′) from the PAM sequence in a gRNA/Cas endonuclease target site, and ii) the 3^(rd) nucleotide in the codon (FIG. 4A). This 3^(rd) position is flexible for most amino acids and can be occupied by any of the four nucleotides in 8 out of 20 amino acids (Table 8). Given such flexibility, a higher frequency of proper repair is anticipated at the 3^(rd) position, when compared to the 1^(st) or 2^(nd) position.

TABLE 8  Genetic code. Compressed Amino Acid Codons Codons Alanine/Ala GCU, GCC, GCA, GCG GCN Arginine/Arg CGU, CGC, CGA, CGG, CGN, MGR AGA, AGG Glycine/Gly GGU, GGC, GGA, GGG GGN Leucine/Leu CUU, CUC, CUA, CUG, CUN, YUR UUA, UUG Proline/Pro CCU, CCC, CCA, CCG CCN Serine/Ser UCU, UCC, UCA, UCG, UCN, AGY AGU, AGC Threonine/Thr ACU, ACC, ACA, ACG ACN Valine/Val GUU, GUC, GUA, GUG GUN Isoleucine/Ile AUU, AUC, AUA AUH Asparagine/Asn AAU, AAC AAY Aspartic Acid/Asp GAU, GAC GAY Cysteine/Cys UGU, UGC UGY Glutamine/Gln CAA, CAG CAR Glutamic Acid/Glu GAA, GAG GAR Histidine/His CAU, CAC CAY Lysine/Lys AAA, AAG AAR Phenylalanine/Phe UUU, UUC UUY Tyrosine/Tyr UAU, UAC UAY Methionine/Met AUG — Tryptophan/Trp UGG —

Four different target sites satisfying the above criteria and the corresponding gRNAs were selected. Besides the above stated preferred single nucleotide position, the corresponding gRNA should promote high frequency of mutations with high percentage of mutations representing a single nucleotide insertion at the cleavage site. Only one of the four target sites tested satisfied all the described preferences and, therefore, suitable for this experiment, was identified (referred to as ALSCas-7; Table 9).

TABLE 9  ALS2-specific ALSCas-7 target site and ALS-CR7- gRNA evaluation by amplicon deep sequencing. % of % of Mutant Mutant Reads Reads % of with with SEQ Target Site Mutant 1 bp 1 bp ID Target Sequence Reads Insertion Deletion NO: ALSCas-4 GCTGCTCGATT 2.73% — — 42 (control) CCGTCCCCA ALSCas-7 GCTCCCCCGGC 2.99% 2.23% 0.14% 79 CACCCCGCTC (75%) (5%)

Then, the ALS2-P165S gene conferring resistance to chlorsulfuron was further modified (resulting in a disrupted gene): the proline codon encoded by CCG (underlined in Table 9) in the ALSCas-7 target site was altered by removal of the G nucleotide at the wobble position (3rd nucleotide position in the codon) resulting in the translational frameshift and a disrupted gene (referred to as ALS2-P165S-CCΔ; FIG. 4 A-4B). As demonstrated in Example 1, repair of DSBs generated by Cas9-gRNA system in maize, and repaired through NHEJ, often results in a single nucleotide insertion at the cleavage site. Therefore, the function of the ALS2-P165S-CCΔ gene, and consecutively, cell resistance to chlorsulfuron, can be restored by generating a double strand break (DSB) at the modified ALSCas-7 site, referred to as ALSCas-7-1 (GCTCCCCCGGCCACCCCCTC; SEQ ID NO: 80) and its repair through NHEJ (see FIG. 4B-4C).

Based on this disclosure, one can envision simultaneous delivery of two or more gRNAs when one gRNA targets and activates the disrupted ALS2-P165S-CCΔ gene through NHEJ (thus conferring herbicide resistance) and the other gRNA(s) promote DSB(s) at a site(s) different than ALS2 and facilitate desired genome modifications, for example, targeted mutagenesis, deletion, gene editing, or site-specific trait gene insertions. This approach can allow for completely transient targeted genome modifications as all other necessary components (Cas9, gRNAs) can be delivered in a form of protein and/or in vitro transcribed RNA molecules.

To test this approach, maize plants (Hi-II genotype) with specifically modified the ALS2 gene described above were generated. First, ALS2 sequence was modified at the amino acid position 165 to confer resistance to chlorsulfuron as described in Example 2. Immature embryos from plants homozygous for the edit were then bombarded with Cas9 and ALS-CR7 gRNA targeting ALSCas-7 target site, selectable marker (UBI:MoPAT-DSRED fusion) and cell developmental enhancing genes (for details, see Examples 8 and 9). Regenerants from bialaphos resistant callus sectors were analyzed by sequencing. Several TO plants with a single nucleotide deletion (a G in the nucleotide position 165) were identified (FIG. 4A-4B). This deletion resulted in the translational frameshift (FIG. 4B) and, hence, loss of ALS2-P165S-mediated chlorsulfuron resistance. Plants homozygous for both edits (ALS2-P165S-CCΔ) were regenerated, confirmed by sequencing and tested for the loss of herbicide resistance by spraying with chlorsulfuron.

Embryos from homozygous plants with specifically modified endogenous ALS2 gene (ALS2-P165S-CCΔ) were used in a prove-of-concept experiment. In order to demonstrate that an edited disrupted ALS2-P165S-CCΔ gene can be repaired as described above (so that it encodes a functional protein) and work as selectable marker, DNA vectors encoding for Cas9, gRNA targeting the ALSCas-7-1 site (referred as ALS-CR7-1), cell development enhancing genes (ZmODP2 and ZmWUS), and MS45-CR2 gRNA were co-delivered into maize (Hi-II) immature embryo cells. One week after bombardment, embryos were transferred to the media with 100 ppm chlorsulfuron for selection. Approximately 30% of embryos (84 out of 290) developed herbicide resistant callus events, which were analyzed by sequencing. The vast majority of the events (79 events) demonstrated a single nucleotide insertion at the expected ALSCas-7-1 DSB site (FIG. 4C) and complete restoration of the ALS2-P165S gene. Four events had no insertion but either 2 or 5 bp deletions putting the gene back in frame, and a single event that seemed to be an escape. Fifty out of 83 events (60%) also demonstrated mutations at the MS45Cas-2 target site.

This example demonstrates prove-of-concept and utility of a specifically modified, inactive ALS2 as endogenous selectable marker gene by the use of a guided Cas endonuclease system. Based on the results described herein, one skilled in the art can use and expand the described approach to any similarly modified endogenous or pre-integrated exogenous gene(s) replacing co-delivery of a selectable marker gene currently used in plant genome editing experiments.

Example 4 Alternative Designs to Restore Function to a Non-Functional Protein Encoded by a Disrupted Gene

In the previous example, sequence alterations were incorporated within the coding region or ALS2-P165S. It is anticipated that others sequence changes which create a disrupted gene (that does not encode a functional protein) can also be designed to be used as re-activation sequences. This example describes generation of a re-activation sequence that does not depend upon the restoration of a codon within a coding sequence, but rather the elimination of a start codon which is upstream and out-of-frame of the primary translational start codon of ALS2-P165S.

According to a scanning model of eukaryotic translation initiation the first AUG codon relative to the 5′ cap of an mRNAs is used to initiate protein synthesis (Kozak M. 1989. The scanning model for translation: an update. The Journal of Cell Biology 108: 229-241). Thus, if an AUG codon within the non-coding leader of the 15 RNA transcript is upstream and out-of-frame of the primary start codon, protein synthesis of the polypeptide encoded by the mRNA is abolished. To take advantage of this rule and apply to a strategy of reactivation of gene expression or function, an endogenous ALS2-P165S allele can be generated to contain an upstream out-of-frame translational start codon (FIG. 5A-5B). This allele contains a Cas9 PAM recognition site and an ALS-CRX targeting spacer. Cutting by Cas9 between nucleotides 3 and 4, located 5′ of the PAM site in this example can promote nucleotide deletion(s) or addition(s) which can result in the loss of the ATG codon. Loss of this upstream out-of-frame ATG by any combination of deletion or addition due to NHEJ repair can result in translation initiation at the primary ALS2-P165S start codon conferring herbicide resistance.

It is anticipated that the re-activation strategy using an upstream out-of-frame ATG not be limited to the design in FIG. 5A-5B. PAM and targeting spacer can also be placed at various positions relative to the upstream out-of-frame ATG, as long as targeted cutting by Cas9 results in the loss of this start codon. For example, the PAM can be present on the antisense strand, 5′ of the start codon. Other designs can be contemplated; the out-of-frame ATG start codon can also be placed at different positions within the 5′ leader sequence. The PAM sequence can be recognized by other Cas9 proteins like Streptococcus pyogenes which recognize nGG PAMs or non-nGG PAM sequences for example Streptococcus thermophiles CR1 (PAM sequence recognition nnAGAAn) and others having PAM sequences. The utility of other Cas9 proteins would satisfy the re-activation design of this example as well as Example 3 described above.

Other designs for gene activation are anticipated. As mentioned earlier, in addition to chlorosulfuron resistance, modifications to the ALS gene which confer resistance to other herbicides can be used for reactivation. Furthermore, the phosphomannose isomerase gene (PMI), bialaphos resistance gene (BAR), phosphinothricin acetyltransferase (PAT), hygromycin resistance gene (NPTII), selectable marker genes, fluorescent marker genes (such as but not limiting to RFP, red fluorescent protein, CFP, GFP, green fluorescent protein) and glyphosate resistance genes can be modified to be introduced into plant cells as inactive forms and used as targets for re-activation by guide RNA introduction and repair by NHEJ as described in Example 3. It would be also anticipated that having multiple inactive genes can serve as targets. For example, guide RNA multiplexing has been demonstrated to simultaneously modify multiple genes in a single experiment, thus targeted reactivation of chlorsulfuron and bialaphos resistance, but not limited to these genes, can be an additionally designed for this approach. As described above, coincident with restoration of gene function by NHEJ, modification of other targets would be accomplished simultaneously by the addition of other guide-polynucleotides.

In addition, it would be anticipated that similar approach can be applied to any native or introduced gene sequence and used as an efficient gene switch mechanism.

Example 5 Specific Gene Editing without Introducing Polynucleotide Modification Templates for Homology Directed Repair

Example 2 described sequence alterations within the coding region of the ALS2 gene (P165S) using specifically designed polynucleotide modification templates (repair templates). The example below describes a different approach to generating an edited gene of interest that does not depend upon the HDR mechanism, but rather on NHEJ.

As described in Example 1, two of the most prevalent types of mutations facilitated by NHEJ repair of DSBs generated by Cas9 nuclease are 1 bp insertion and 1 bp deletion (Table 4). Based on these observations described herein, methods were developed for gene editing that can be accomplished in two consecutive steps or into a single step, as described below.

The first step includes targeting a gene or polynucleotide of interest, containing a target site that is recognized by a Cas endonuclease, using the RNA guided Cas nuclease system described herein, resulting in the generation of a cell or an organism with a specific nucleotide deletion due to NHEJ repair of the cleaved DNA (illustrated in FIG. 6A). The second step requires re-targeting the mutated site and selecting events with insertion of a desired nucleotide (without the use of a polynucleotide modification template (repair template), hence, specifically changing the corresponding amino acid and the gene function. In general, the idea is illustrated in FIG. 6A-6C. This method can also be used to edit non-coding DNA fragments.

Alternatively, both steps can be combined into a single step. Two different gRNAs, one recognizing the original target site and the second one the same site but with a 1 bp deletion can be used. In this case, one can envision a consecutive cutting and repair of the endogenous site resulting in a 1 bp deletion followed by cutting of the altered site and its repair with a 1 bp insertion. Then, an event with an insertion of a desired nucleotide can be selected. In the case of editing coding DNA sequences, this process accomplishes two goals—restoring the translational reading frame and replacement of an amino acid of interest. It would be anticipated that combinations of different endonuclease could be used in this two-step or one-step system. For example, the introduction of a first double strand break leading to a single base deletion in a polynucleotide of interest can be accomplished by a first endonuclease, whereas the introduction of a second double strand break leading to a single base insertion (and editing of the polynucleotide of interest) can be accomplished by a second endonuclease that is different from the first endonuclease. The differences between the endonucleases may include, but are not limited to, different PAM recognition sequences, different target recognition sequences, different cleavage activity (blunt-end, 5′ or 3′ overhang, single strand, double strand), different DNA or amino acid sequences, originating from different organism, or any one combination thereof.

The ability to edit a specific nucleotide in a genome of interest may depend on the endonuclease system of choice and its ability to recognize and cut a particular target site. The discovery of novel guided endonucleases (See for example U.S. patent application 62/162,377 filed May 15 2015), and/or modifications of guided endonucleases with various PAM sequences, will further increase the density of target sites that can be recognized and/or cleaved by these endonuclease ultimately resulting in the ability to target any given nucleotide position in the genome using the methods described herein.

Example 6 Maize Lines with Stably Integrated Cas9 Endonuclease

This example describes generation and validation of maize lines with stably integrated Cas9 expression cassette.

Two Agrobacterium vectors (FIG. 7, containing maize-codon optimized Cas9 under the transcriptional control of a constitutive (maize UBI, SEQ ID NO: 46) or a temperature regulated (maize MDH, SEQ ID NO: 47) promoter were introduced into Hi-II embryo cells to establish lines containing pre-integrated genomic copies of Cas9 endonuclease. These vectors also contained an embryo-preferred END2 promoter regulating the expression of a blue-fluorescence gene (AmCYAN) as a visible marker and an interrupted copy of the DsRED gene transcriptionally regulated by a maize Histone 2B promoter. Part of the DsRED sequence was duplicated in a direct orientation (369 bp fragment) and consisted of two fragments of the DsRED (RF-FP) gene which were separated by a 347 bp spacer that could be targeted by gRNAs. DSBs within the spacer region promote intramolecular recombination restoring function to the disrupted DsRED gene which results in red fluorescing cells. Maize plants with single-copy T-DNA inserts containing either UBI:Cas9 or MDH:Cas9 were used as a source of immature embryos. Blue-fluorescing embryos containing pre-integrated Cas9 were excised and incubated at 28° C. (UBI:Cas9) or at 37° C. (MDH:Cas9) for 24 hours. Post-bombardment, embryos with MDH:Cas9 were incubated at 37° C. for 24 hours and then moved to 28° C. In contrast to control (no gRNA), UBI:Cas9 and MDH:Cas9 containing embryos bombarded with two DNA-expressed gRNAs that targeted sequences within the 347 bp spacer, readily produced red fluorescing foci.

These results demonstrate that described above maize lines poses single copies of functional Cas9 endonuclease.

Example 7 Transient gRNA Delivery into Embryo Cells with Pre-Integrated Cas9 Generates Mutations in Maize

This Example demonstrates that delivery of gRNA in a form of in vitro transcribed RNA molecules into maize immature embryo cells with pre-integrated Cas 9 generates mutations at targeted sites.

Maize plants described in Example 6 containing either UBI:Cas9 or MDH:Cas9 were used as a source of immature embryos for delivery of gRNAs as in vitro transcribed RNA or as DNA expression cassettes as control. To measure mutation frequencies at the LIG and MS26 endogenous target sites, LIG-CR3 and MS26-CR2 gRNAs as RNA molecules (100 ng/shot) or as DNA vectors (25 ng/shot) were delivered into UBI:Cas9 and MDH:Cas9 containing embryo cells with temperature treatments described in Example 6. In these experiments, embryos were harvested two days post-bombardment and analyzed by amplicon deep sequencing. Similar frequencies were detected for gRNAs delivered as DNA vectors and as RNA molecules, particularly in the case of Cas9 regulated by the Ubiquitin promoter (Table 10).

TABLE 10 Percentage of mutant reads at maize LIG and MS26 target sites produced by transient gRNA delivery into embryos with pre-integrated Cas9 under constitutive (UBI) or regulated (MDH) promoters Percentage of Mutant Reads Target Site Embryos Transformation (2 days post-bombardment) LIG UBI:Cas9 gRNA (DNA) 1.22% gRNA (RNA) 1.86% MDH:Cas9 gRNA (DNA) 0.25% event 1 gRNA (RNA) 0.12% MDH:Cas9 gRNA (DNA) 0.57% event 2 gRNA (RNA) 0.26% MDH:Cas9 gRNA (DNA) 0.46% event 3 gRNA (RNA) 0.35% MS26 MDH:Cas9 gRNA (DNA) 0.58% event 2 gRNA (RNA) 0.17%

Together, these data demonstrate that delivery of gRNA in the form of RNA directly into maize cells containing pre-integrated Cas9 is a viable alternative to DNA delivery for the generation of mutations in plant cells.

Example 8 Transformation of Maize Immature Embryos

Transformation can be accomplished by various methods known to be effective in plants, including particle-mediated delivery, Agrobacterium-mediated transformation, PEG-mediated delivery, and electroporation.

a. Particle-Mediated Delivery

Transformation of maize immature embryos using particle delivery is performed as follows. Media recipes follow below.

The ears are husked and surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are isolated and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5-cm target zone in preparation for bombardment. Alternatively, isolated embryos are placed on 560L (Initiation medium) and placed in the dark at temperatures ranging from 26° C. to 37° C. for 8 to 24 hours prior to placing on 560Y for 4 hours at 26° C. prior to bombardment as described above.

Plasmids containing the double strand brake inducing agent and template or donor DNA are constructed using standard molecular biology techniques and co-bombarded with plasmids containing the developmental genes ODP2 (AP2 domain transcription factor ODP2 (Ovule development protein 2); US20090328252 A1) and WUSCHEL (US2011/0167516).

The plasmids and DNA of interest are precipitated onto 0.6 μm (average diameter) gold pellets using a water-soluble cationic lipid TransIT-2020 Transfection Reagent (Cat# MIR 5404, Mirus, USA) as follows. DNA or DNA and RNA solution is prepared on ice using a total of 1 μg of DNA and/or RNA constructs (10 shots). To the pre-mixed DNA, 20 μl of prepared gold particles (15 mg/ml) and 1 μl TransIT-2020 are added and mixed carefully. Gold particles are pelleted in a microfuge at 10,000 rpm for 1 min and supernatant is removed. 105 μl of 100% EtOH is added and the particles are resuspended by brief sonication. Then, 10 μl is spotted onto the center of each macrocarrier and allowed to dry before bombardment. The sample plates are bombarded using Biorad Helium Gun (shelf #3) at 425 PSI.

Following bombardment, the embryos are incubated on 560P (maintenance medium) for 12 to 48 hours at temperatures ranging from 26 C to 37 C, and then placed at 26 C. After 5 to 7 days the embryos are transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks at 26 C. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to a lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to a 2.5″ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to Classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for transformation efficiency, and/or modification of regenerative capabilities.

Initiation medium (560L) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 20.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).

Maintenance medium (560P) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, 2.0 mg/l 2,4-D, and 0.69 g/l L-proline (brought to volume with D-I H₂O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).

Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H2O after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.).

Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H2O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H2O), sterilized and cooled to 60° C.

b. Agrobacterium-Mediated Transformation

Agrobacterium-mediated transformation was performed essentially as described in Djukanovic et al. (2006) Plant Biotech J 4:345-57. Briefly, 10-12 day old immature embryos (0.8-2.5 mm in size) were dissected from sterilized kernels and placed into liquid medium (4.0 g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/L thiamine HCl, 1.5 mg/L 2, 4-D, 0.690 g/L L-proline, 68.5 g/L sucrose, 36.0 g/L glucose, pH 5.2). After embryo collection, the medium was replaced with 1 ml Agrobacterium at a concentration of 0.35-0.45 OD550. Maize embryos were incubated with Agrobacterium for 5 min at room temperature, then the mixture was poured onto a media plate containing 4.0 g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/L thiamine HCl, 1.5 mg/L 2, 4-D, 0.690 g/L L-proline, 30.0 g/L sucrose, 0.85 mg/L silver nitrate, 0.1 nM acetosyringone, and 3.0 g/L Gelrite, pH 5.8. Embryos were incubated axis down, in the dark for 3 days at 20° C., then incubated 4 days in the dark at 28° C., then transferred onto new media plates containing 4.0 g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/L thiamine HCl, 1.5 mg/L 2, 4-D, 0.69 g/L L-proline, 30.0 g/L sucrose, 0.5 g/L MES buffer, 0.85 mg/L silver nitrate, 3.0 mg/L Bialaphos, 100 mg/L carbenicillin, and 6.0 g/L agar, pH 5.8. Embryos were subcultured every three weeks until transgenic events were identified. Somatic embryogenesis was induced by transferring a small amount of tissue onto regeneration medium (4.3 g/L MS salts (Gibco 11117), 5.0 ml/L MS Vitamins Stock Solution, 100 mg/L myo-inositol, 0.1 μM ABA, 1 mg/L IAA, 0.5 mg/L zeatin, 60.0 g/L sucrose, 1.5 mg/L Bialaphos, 100 mg/L carbenicillin, 3.0 g/L Gelrite, pH 5.6) and incubation in the dark for two weeks at 28° C. All material with visible shoots and roots were transferred onto media containing 4.3 g/L MS salts (Gibco 11117), 5.0 ml/L MS Vitamins Stock Solution, 100 mg/L myo-inositol, 40.0 g/L sucrose, 1.5 g/L Gelrite, pH 5.6, and incubated under artificial light at 28° C. One week later, plantlets were moved into glass tubes containing the same medium and grown until they were sampled and/or transplanted into soil.

Example 9 Transient Expression of ZmODP-2 and ZmWUS Enhances Transformation

Parameters of the transformation protocol can be modified to ensure that the BBM activity is transient. One such method involves precipitating the BBM-containing plasmid in a manner that allows for transcription and expression, but precludes subsequent release of the DNA, for example, by using the chemical PEI.

In one example, the BBM plasmid is precipitated onto gold particles with PEI, while the transgenic expression cassette (UBI:MoPAT-GFPm:PinII; MoPAT is the maize optimized PAT gene) to be integrated is precipitated onto gold particles using the standard calcium chloride method.

Briefly, gold particles were coated with PEI as follows. First, the gold particles were washed. Thirty-five mg of gold particles, 1.0 in average diameter (A.S.I. #162-0010), were weighed out in a microcentrifuge tube, and 1.2 ml absolute EtOH was added and vortexed for one minute. The tube was incubated for 15 minutes at room temperature and then centrifuged at high speed using a microfuge for 15 minutes at 4° C. The supernatant was discarded and a fresh 1.2 ml aliquot of ethanol (EtOH) was added, vortexed for one minute, centrifuged for one minute, and the supernatant again discarded (this is repeated twice). A fresh 1.2 ml aliquot of EtOH was added, and this suspension (gold particles in EtOH) was stored at −20° C. for weeks. To coat particles with polyethylimine (PEI; Sigma #P3143), 250 μl of the washed gold particle/EtOH mix was centrifuged and the EtOH discarded. The particles were washed once in 100 μl ddH2O to remove residual ethanol, 250 μl of 0.25 mM PEI was added, followed by a pulse-sonication to suspend the particles and then the tube was plunged into a dry ice/EtOH bath to flash-freeze the suspension, which was then lyophilized overnight. At this point, dry, coated particles could be stored at −80° C. for at least 3 weeks. Before use, the particles were rinsed 3 times with 250 μl aliquots of 2.5 mM HEPES buffer, pH 7.1, with 1× pulse-sonication, and then a quick vortex before each centrifugation. The particles were then suspended in a final volume of 250 μl HEPES buffer. A 25 μl aliquot of the particles was added to fresh tubes before attaching DNA. To attach uncoated DNA, the particles were pulse-sonicated, then 1 μg of DNA (in 5 μl water) was added, followed by mixing by pipetting up and down a few times with a Pipetteman and incubated for 10 minutes. The particles were spun briefly (i.e. 10 seconds), the supernatant removed, and 60 μl EtOH added. The particles with PEI-precipitated DNA-1 were washed twice in 60 μl of EtOH. The particles were centrifuged, the supernatant discarded, and the particles were resuspended in 45 μl water. To attach the second DNA (DNA-2), precipitation using TransIT-2020 was used. The 45 μl of particles/DNA-1 suspension was briefly sonicated, and then 5 μl of 100 ng/μl of DNA-2 and 1 μl of TransIT-2020 were added. The solution was placed on a rotary shaker for 10 minutes, centrifuged at 10,000 g for 1 minute. The supernatant was removed, and the particles resuspended in 60 μl of EtOH. The solution was spotted onto macrocarriers and the gold particles onto which DNA-1 and DNA-2 had been sequentially attached were delivered into scutellar cells of 10 DAP Hi-II immature embryos using a standard protocol for the PDS-1000. For this experiment, the DNA-1 plasmid contained a UBI:RFP:PinII expression cassette, and DNA-2 contained a UBI:CFP:PinII expression cassette. Two days after bombardment, transient expression of both the CFP and RFP fluorescent markers was observed as numerous red & blue cells on the surface of the immature embryo. The embryos were then placed on non-selective culture medium and allowed to grow for 3 weeks before scoring for stable colonies. After this 3-week period, 10 multicellular, stably-expressing blue colonies were observed, in comparison to only one red colony. This demonstrated that PEI-precipitation could be used to effectively introduce DNA for transient expression while dramatically reducing integration of the PEI-introduced DNA and thus reducing the recovery of RFP-expressing transgenic events. In this manner, PEI-precipitation can be used to deliver transient expression of BBM and/or WUS2.

For example, the particles are first coated with UBI:BBM:PinII using PEI, then coated with UBI:MoPAT-YFP using TransIT-2020, and then bombarded into scutellar cells on the surface of immature embryos. PEI-mediated precipitation results in a high frequency of transiently expressing cells on the surface of the immature embryo and extremely low frequencies of recovery of stable transformants (relative to the TransIT-2020 method). Thus, it is expected that the PEI-precipitated BBM cassette expresses transiently and stimulates a burst of embryogenic growth on the bombarded surface of the tissue (i.e. the scutellar surface), but this plasmid will not integrate. The MoPAT-GFP plasmid released from the Ca++/gold particles is expected to integrate and express the selectable marker at a frequency that results in substantially improved recovery of transgenic events. As a control treatment, PEI-precipitated particles containing a UBI:GUS:PinII (instead of BBM) are mixed with the MoPAT-GFP/Ca++ particles. Immature embryos from both treatments are moved onto culture medium containing 3 mg/l bialaphos. After 6-8 weeks, it is expected that GFP+, bialaphos-resistant calli will be observed in the PEI/BBM treatment at a much higher frequency relative to the control treatment (PEI/GUS).

As an alternative method, the BBM plasmid is precipitated onto gold particles with PEI, and then introduced into scutellar cells on the surface of immature embryos, and subsequent transient expression of the BBM gene elicits a rapid proliferation of embryogenic growth. During this period of induced growth, the explants are treated with Agrobacterium using standard methods for maize (see Example 1), with T-DNA delivery into the cell introducing a transgenic expression cassette such as UBI:MoPAT-GFPm:PinII. After co-cultivation, explants are allowed to recover on normal culture medium, and then are moved onto culture medium containing 3 mg/l bialaphos. After 6-8 weeks, it is expected that GFP+, bialaphos-resistant calli will be observed in the PEI/BBM treatment at a much higher frequency relative to the control treatment (PEI/GUS).

It may be desirable to “kick start” callus growth by transiently expressing the BBM and/or WUS2 polynucleotide products. This can be done by delivering BBM and WUS2 5′-capped polyadenylated RNA, expression cassettes containing BBM and WUS2 DNA, or BBM and/or WUS2 proteins. All of these molecules can be delivered using a biolistics particle gun. For example 5′-capped polyadenylated BBM and/or WUS2 RNA can easily be made in vitro using Ambion's mMessage mMachine kit. RNA is co-delivered along with DNA containing a polynucleotide of interest and a marker used for selection/screening such as UBI:MoPAT-GFPm:PinII. It is expected that the cells receiving the RNA will immediately begin dividing more rapidly and a large portion of these will have integrated the agronomic gene. These events can further be validated as being transgenic clonal colonies because they will also express the PAT-GFP fusion protein (and thus will display green fluorescence under appropriate illumination). Plants regenerated from these embryos can then be screened for the presence of the polynucleotide of interest.

Example 10 Direct Delivery of gRNA and Cas9 as a Guide RNA/Cas Endonuclease Ribonucleotide-Protein Complex (RGEN) into Embryo Cells Generates Mutations in Maize

This example demonstrates that direct delivery of Cas9 in the form of protein and gRNA in the form of in vitro transcribed or chemically synthesized RNA molecules, into maize immature embryo cells generates mutations at the corresponding targeted sites.

To generate gRNA in the form of RNA molecules, the maize-optimized U6 polymerase III gRNA expression cassettes were amplified by PCR using a 5′ oligonucleotide primer that also contained the sequence of the T7 polymerase promoter and transcriptional initiation signal just 5′ of the spacer to gene. T7 in vitro transcription was carried out with the AmpliScribe T7-Flash Kit (Epicentre) according to the manufacturer's recommendations, and products were purified using NucAway Spin Columns (Invitrogen; Life Technologies Inc) followed by ethanol precipitation.

To generate a guide RNA/Cas9 endonuclease protein complex (RGEN) (also referred to as a guide RNA/Cas9 endonuclease ribonucleotide-protein), 7 μg of Cas9 (Streptococcus pyogenes Cas9) protein and 3 μg of gRNA molecules (1:2 molar ratio) were mixed in 1× Cas9 buffer (NEB) in a total volume of 20 μl and incubated at room temperature for 15 minutes. Together with the RGEN, plasmids containing Ubiquitin promoter regulated selectable and visible markers (MoPAT-DsRed fusion), Ubiquitin promoter regulated mays Ovule development protein 2, ZmODP2 (see US20090328252, published Dec. 31, 2009) and maize IN2 promoter (Hershey et al. 1991, Plant Mol. Biol 17:679-690) regulated WUSCHEL, ZmWUS (see US20110167516, published Jul. 7, 2011) were mixed with a particle delivery matrix comprising commercially available gold particles (0.6 μm, Bio-Rad) and a water soluble cationic lipid TransIT-2020 (Mirus, USA). The particle delivery matrix comprising the guide RNA/Cas endonuclease ribonucleotide-protein complexes were delivered into maize embryo cells using particle mediated delivery (see Particle-mediated delivery described in Example 8) with some modifications. Specifically, after gold particles were pelleted in a microfuge at 10,000 rpm for 1 min and supernatant was removed, the particles were resuspended in 105 μl of sterile water instead of 100% ethanol. Then, 10 μl was spotted onto the center of each macrocarrier and allowed to dry before bombardment.

Wild type maize plants were used as a source of immature embryos for co-delivery of Cas9 and gRNA in the form of a guide RNA/Cas endonuclease ribonucleotide-protein complexes (RGEN) along with selectable and visible marker (UBI:MoPAT-DsRED) and developmental genes (UBI:ZmODP2 and IN2:ZmWUS). To measure mutation frequencies at the LIGCas-3, MS26Cas-2, MS45Cas-2 and ALSCas-4 endogenous target sites, embryos were harvested two days post-bombardment and analyzed by amplicon deep sequencing. Untreated embryos and embryos bombarded with the Cas9 protein only served as negative controls while embryos bombarded with DNA vectors expressing Cas9 and gRNA were used as positive controls. Similar frequencies were detected for Cas9-gRNA components delivered as DNA vectors and as guide RNA/Cas endonuclease ribonucleotide-protein complexes (Table 11).

TABLE 11 Percentage of mutant reads at LIG, MS26, MS45, and ALS target sites produced by direct delivery of RGEN complexes into maize embryo cells. Total Number Percentage Target Number of of Mutant of Mutant Sites Molecules delivered Reads Reads Reads LIGCas-3 Untreated embryos 915,198 38 0.004% (Chr. 2) (control 1) Cas9 protein only 408,348 17 0.004% (control 2) Cas9-Lig-CR3 439,827 2,510 0.57% RGEN Cas9 (DNA) + 369,443 2,058 0.56% Lig-CR3 (DNA) MS26Cas-2 Untreated embryos 245,476 8 0.003% (Chr. 1) (control 1) Cas9 protein only 429,388 20 0.004% (control 2) Cas9-MS26-CR2 252,519 533 0.21% RGEN Cas9 (DNA) + 186,857 812 0.43% MS26-CR2 (DNA) MS45Cas-2 Untreated embryos 255,877 12 0.005% (Chr. 9) (control 1) Cas9 protein only 487,876 12 0.002% (control 2) Cas9-MS45-CR2 241,287 2,075 0.86% RGEN Cas9 (DNA) + 304,622 1591 0.52% MS45-CR2 (DNA) ALS2Cas-4 Cas9 protein only 807,014 125 0.02% (Chr. 5) (control 2) Cas9-ALS-CR4 791,084 3,613 0.45% RGEN Cas9 (DNA) + 833,130 4251 0.51% ALS-CR4 (DNA)

To measure the mutation frequency at the plant level, 60 embryos co-bombarded with Cas9-MS45-gRNA complex, ZmODP2, ZmWUS and MOPAT-DSRED were placed on media containing bialaphos as selective agent. Multiple plants were regenerated from each of the 36 herbicide-resistant callus sectors and screened for mutations. Out of the 36 events, 17 (47%) contained mutant alleles (10 single and 7 biallelic) while 19 (53%) revealed only wild type MS45 alleles. Among plants with mutations, the number of sequencing reads for each allele was similar indicating plants were not chimeric.

To demonstrate that direct RGEN delivery is also sufficient to generate specific edits in endogenous genes in plants, the maize ALS2 gene was targeted (ALS2-specific ALSCas-4 target site) as described in Example 2. A 127 nt single-stranded DNA Oligo2, (SEQ ID NO: 45) as a repair template was co-delivered with Cas9/ALS-CR4 RGEN complex in a similar manner as described above. Embryos were harvested two days post-bombardment and analyzed by amplicon deep sequencing (Table 12).

TABLE 12 Percentage of mutant reads and reads with edits at ALS target site produced by delivery of RGEN complex and donor DNA template into maize embryo cells. Total Number of % of Number of % of Target Molecules Number of Mutant Mutant Reads with Reads with Site delivered Reads Reads Reads Edits Edits ALS2 Cas9 protein 807,014 105 0.01% — — only Cas9-ALS- 791,084 3,613 0.45% 209 0.02% CR4 RGEN + ss Oligo2

In addition, in two independent experiments, 40 to 50 bombarded embryos were transferred to plates containing 100 ppm of chlorsulfuron as direct selection for an edited ALS2 gene. Six weeks later, two callus sectors (one from each experiment) that continued growing on media with chlorsulfuron were analyzed by sequencing. In both events, one ALS2 allele was specifically edited while the second allele remained wild type. Plants regenerated from these callus sectors contained edited ALS2 alleles and were resistant to chlorsulfuron when sprayed with the herbicide.

These data demonstrate that direct delivery of Cas9 and gRNA, in the form of a guide RNA/Cas endonuclease ribonucleotide-protein complex (with or without a polynucleotide modification template DNA) into maize immature embryo cells, is a viable alternative to DNA delivery (such as recombinant DNA, plasmid DNA) for targeted mutagenesis and gene editing in plants.

Example 11 Direct Delivery of Cas9 in the Form on mRNA and gRNA into Embryo Cells Generates Mutations in Maize

This example demonstrates that direct delivery of Cas9, in the form of mRNA molecules and gRNA in the form of in vitro transcribed or chemically synthesized RNA molecules, into maize immature embryo cells generates mutations at the corresponding targeted sites.

In our previous experiment (Svitashev et al., Plant Physiology, 2015, Vol. 169, pp. 931-945), co-delivery of gRNA in the form of in vitro synthesized RNA molecules with the Cas9 expressing DNA vector yielded approximately 100-fold lower mutation frequency then in experiments where both Cas9 and gRNA were delivered as DNA vectors. One possible explanation for this difference may be that the requirement for coincident function of Cas9 and gRNA was not met when gRNA was delivered as RNA and Cas9 was delivered as a DNA vector. To overcome this problem, Cas9 can be delivered as mRNA molecules that will shorten the time from the moment of delivery to functional Cas9 protein expression. Commercially available Cas9 m RNA (TriLink Biotechnologies) was used in the experiment.

To test this idea, maize embryo cells were co-bombarded with Cas9 mRNA (200 ng), gRNA in the form of in vitro synthesized RNA molecules (100 ng), DNA vectors containing Ubiquitin regulated MoPAT-DsRED fusion (25 ng) and developmental genes, Ubiquitin promoter regulated ODP2 and IN2 promoter regulated WUS (12 ng each) per shot. Commercially available Cas9 mRNA (TriLink Biotechnologies) and RNA molecules, in vitro synthesized as described above, were used in the experiment. Analysis for mutation frequency was performed by amplicon deep sequencing on embryos collected 2 days post-transformation (Table 13).

TABLE 13 Percentage of mutant reads at MS45 target site produced by transient delivery of Cas9 as mRNA and gRNA as RNA molecules into maize embryo cells. Total Number Percentage Target Number of Mutant of Mutant Sites Molecules delivered of Reads Reads Reads MS45 Cas9 mRNA only 1,097,279 799 0.07% Cas9 mRNA + 1,260,332 2,304 0.18% Ms45 CR2 gRNA Cas9 (DNA) + 1,106,125 3,490 0.31% Ms45 CR2 (DNA)

These data demonstrate that delivery of both Cas9 and gRNA, in the form of RNA molecules, improves frequencies of targeted mutations and, along with Cas9-gRNA delivery as the RGEN complex, is a viable alternative to DNA delivery for targeted mutagenesis and gene editing in plants.

Example 12 Direct Delivery of Cas9 and gRNA as a Guide RNA/Cas Endonuclease Ribonucleotide-Protein Complex (RGEN) into Embryo Cells without the Use of a Selectable Marker Generates Mutations in Maize

This example demonstrates that delivery of Cas9 in the form of protein and gRNA in the form of in vitro transcribed or chemically synthesized RNA molecules, into maize immature embryo cells without co-delivery of selectable marker gene(s) is sufficient to regenerate plants with mutations at the corresponding targeted sites with practical frequencies.

The necessity of selectable markers to provide a growth advantage to transformed or modified cells has been the long standing paradigm in plant transformation and genome modification protocols. Therefore, in all mutation, gene editing and gene integration experiments, selectable markers are used to select for genome edited events. Taking into consideration the unexpected high activity (mutation frequency) of RGEN complexes in the experiments described in Example 10, a completely DNA-free (vector free) genome editing without a selectable marker was attempted. Maize embryo cells were bombarded with guide RNA/Cas endonuclease ribonucleotide-protein (RGEN) complexes targeting three different genes: liguleless1 (LIG), MS26 and MS45. Cas9 endonuclease and MS45-gRNA on DNA vectors were delivered in parallel experiments which served as controls. Plants were regenerated and analyzed by sequencing for targeted mutations. In all experiments, mutant plants were recovered at surprisingly high frequencies ranging from 2.4 to 9.7% (Table 14).

TABLE 14 Mutation frequencies at LIG, MS26 and MS45 target sites upon delivery of Cas9 and gRNAs in the form of DNA vectors and direct delivery of RGEN complexes into maize immature embryo cells. Analysis was performed on T0 plants regenerated without selection (without use of selectable markers). Cas9 and gRNA Plants Plants with SEQ Target site delivery method analyzed mutated alleles ID NO: LIGCas-3 RGEN 756 73 (9.7%) 12 MS26Cas-2 RGEN 756 18 (2.4%) 14 MS45 Cas-2 RGEN 1,880 70 (3.7%) 8 MS45 Cas-2 Vector DNA 940 38 (4.0%) 8

Further, regenerated T0 plants were crossed with wild type Hi-II plants and the progeny plants were used for segregation analysis. Sexual transmission of mutated ms45 alleles at the expected Mendelian segregation (1:1) was demonstrated in all progeny plants analyzed.

To assess the potential of RGEN delivery to reduce off-target cleavage in maize, the mutation frequency at the MS45 off-site was evaluated using DNA vectors and RGEN delivery. Searches for a site with close homology to the on-target site were performed by aligning the protospacer region of the MS45Cas-2 target site (the region of the target site that base pairs with the guide RNA spacer) with the maize B73 reference genome (B73 RefGen_v3, Maize Genetics and Genomics Database) using Bowtie sequence aligner (Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10:R25, 2009) permitting up to two mismatches with the on-target sequence. Potential off-target sites were then examined for the presence of a NGG protospacer adjacent motif (PAM) sequence immediately 3′ of the identified protospacer off-target. Only a single off-target site (5′-CGCCGAGGGCGACTACCGGC-3′, SEQ ID NO:81) was identified using these search criteria. It contained a 2 bp mismatch with the MS45 protospacer target and an AGG PAM (Table 15). To confirm the site was cleaved in vivo, it was analyzed by deep sequencing for the presence of mutations in maize embryos transformed with DNA vectors expressing Cas9 and MS45-CR2 gRNA. As shown in Table 15, mutational activity of the off-target site was at a frequency of 2% compared to a 4% frequency observed for the on-target site. As shown in Table 15, RGEN off-target activity was greatly reduced relative to Cas9 and gRNA delivery on DNA vectors (from 2% to 0%).

TABLE 15  Mutation frequencies at the MS45 off-target site upon delivery of Cas9 and gRNAs in the form of DNA vectors and RGEN complexes into maize immature embryo cells. Analysis performed on T0 plants regenerated without selection. Cas9 Target Plants Plants and site with with gRNA sequence Muta- Muta- SEQ Target  delivery with Plants tions tions ID Site method PAM* Analyzed (number) (%) NO: MS45Cas- GGCCGAGG   904 38   4% 14 2 TCGACTAC CGGCCGG M545 DNA C GCCGAGG   940 19 2.0% 82 off-site G CGACTAC CGGC AGG MS45 RGEN C GCCGAGG 1,880  0 0.0% 82 off-site G CGACTAC CGGC AGG *PAM-protospacer adjacent motif is a 3 nt sequence immediately 3′ of the target site. Two nucleotides different in the MS45 off-target site in comparison to the intended site are shown in bold and underlined.

This example demonstrates that generation of plants with targeted mutations using RNA-guided endonucleases, does not require co-delivery of selectable or screenable marker genes, thus increasing specificity and exactness of the introduced modifications. Regenerated plants contained only targeted mutations or targeted gene edits (if a repair template was included to modify DNA sequence) without random integration of DNA vectors. This method of delivery provides a completely DNA-free approach to gene mutagenesis in plant cells of major crop species including but not limited to maize, soybean, wheat, rice, millet, sorghum and canola. 

1.-24. (canceled)
 25. A method of introducing a Cas9 protein and a guide RNA into a plant cell, said method comprising: introducing into the plant cell the Cas9 protein and the guide RNA, wherein the guide RNA comprises a polynucleotide sequence complementary to a genomic target site sequence in the plant cell; wherein the Cas9 protein and the guide RNA are applied to a microparticle; and wherein the Cas9 protein and the guide RNA bind to the genomic target site sequence in the plant cell.
 26. The method of claim 25, wherein the Cas9 protein and the guide RNA are introduced concurrently.
 27. The method of claim 25, wherein the Cas9 protein and the guide RNA are introduced separately.
 28. The method of claim 25, wherein the Cas9 protein and the guide RNA are introduced as a ribonucleoprotein complex.
 29. The method of claim 25, wherein the plant cell is selected from the group consisting of: maize, soybean, cotton, canola, wheat, rice, Arabidopsis, alfalfa, tobacco, and sorghum.
 30. A method of modifying a target sequence in a plant cell, the method comprising: a. introducing into the plant cell a microparticle coated with a ribonucleoprotein complex comprising a Cas9 protein and a guide RNA, and b. incubating the plant cell to allow the ribonucleoprotein complex to bind to the target sequence; wherein the guide RNA molecule comprises a polynucleotide sequence complementary to a genomic target site sequence in the plant cell; and wherein the target sequence is modified by the insertion of at least one nucleotide, the deletion of at least one nucleotide, or the substitution of at least one nucleotide.
 31. The method of claim 30, wherein the plant cell is selected from the group consisting of: maize, soybean, cotton, canola, wheat, rice, Arabidopsis, alfalfa, tobacco, and sorghum.
 32. The method of claim 30, wherein the Cas9 protein is catalytically inactive.
 33. The method of claim 30, further comprising introducing into the plant cell a polynucleotide modification template, wherein said polynucleotide modification template comprises at least one nucleotide modification as compared to the target site sequence.
 34. The method of claim 30, further comprising introducing into the plant a donor DNA molecule, wherein the donor DNA molecule comprises at least one heterologous polynucleotide of interest to be inserted into said target site.
 35. A composition comprising a plant cell and a particle delivery matrix, wherein the particle delivery matrix comprises: a. a microparticle, b. a Cas9 protein, and c. a guide RNA molecule comprising a polynucleotide sequence complementary to a genomic target site sequence; wherein the microparticle is associated with the Cas9 protein and the guide RNA molecule.
 36. The composition of claim 35, wherein the Cas9 protein and the guide RNA form a complex that binds to a target site in the plant cell.
 37. The composition of claim 35, wherein the plant cell is selected from the group consisting of: maize, soybean, cotton, canola, wheat, rice, Arabidopsis, alfalfa, tobacco, and sorghum.
 38. The composition of claim 35, wherein the Cas9 protein is catalytically inactive.
 39. The composition of claim 35, further comprising a heterologous polynucleotide.
 40. The composition of claim 38, wherein the heterologous polynucleotide is a polynucleotide modification template, wherein the polynucleotide modification template comprises at least one nucleotide modification as compared to the target site sequence.
 41. The composition of claim 38, wherein the heterologous polynucleotide is a donor DNA molecule, wherein the donor DNA molecule comprises at least one heterologous polynucleotide of interest to be inserted into said target site. 